Bone Organoids: Recent Advances and Future Challenges
骨类器官：最新进展和未来挑战
Fulltext @SJTU 全文@SJTUAbstract 抽象
Bone defects stemming from tumorous growths, traumatic events, and diverse conditions present a profound conundrum in clinical practice and research. While bone has the inherent ability to regenerate, substantial bone anomalies require bone regeneration techniques. Bone organoids represent a new concept in this field, involving the 3D self-assembly of bone-associated stem cells guided in vitro with or without extracellular matrix material, resulting in a tissue that mimics the structural, functional, and genetic properties of native bone tissue. Within the scientific panorama, bone organoids ascend to an esteemed status, securing significant experimental endorsement. Through a synthesis of current literature and pioneering studies, this review offers a comprehensive survey of the bone organoid paradigm, delves into the quintessential architecture and ontogeny of bone, and highlights the latest progress in bone organoid fabrication. Further, existing challenges and prospective directions for future research are identified, advocating for interdisciplinary collaboration to fully harness the potential of this burgeoning domain. Conclusively, as bone organoid technology continues to mature, its implications for both clinical and research landscapes are poised to be profound.
由肿瘤生长、创伤事件和各种情况引起的骨缺损在临床实践和研究中是一个深刻的难题。虽然骨骼具有固有的再生能力，但大量的骨骼异常需要骨骼再生技术。骨类器官代表了该领域的一个新概念，涉及在体外引导下使用或不使用细胞外基质材料的骨相关干细胞的 3D 自组装，从而产生模拟天然骨组织的结构、功能和遗传特性的组织。在科学全景中，骨类器官上升到受人尊敬的地位，获得了重要的实验认可。通过综合当前文献和开创性研究，本文对骨类器官范式进行了全面调查，深入探讨了骨骼的典型结构和个体发育，并强调了骨类器官制造的最新进展。此外，确定了现有挑战和未来研究的前瞻性方向，倡导跨学科合作以充分利用这一新兴领域的潜力。总而言之，随着骨类器官技术的不断成熟，其对临床和研究领域的影响将是深远的。
1 Introduction 1 引言
Bone defects, tumors, and other bone-related diseases have been major health concerns for a long time. The protracted recovery timelines associated with bone and joint injuries not only jeopardize patient well-being but also impose considerable economic ramifications. Osteoarthritis, for instance, is a disease that affects millions of people worldwide, with a staggering 303 million people suffering from knee and hip osteoarthritis. Given these challenges, there is a pressing need for a better understanding of bone development, regulation, disease mechanisms, progression, and drug screening. Currently, traditional laboratory research methods rely on 2D cell culture and animal experiments. However, these methods have significant limitations, including a lack of spatial structure, extracellular matrix, and cell-environment interactions under physiological conditions. As such, they cannot fully mimic the complex and heterogeneous conditions found in vivo. To overcome these limitations, 3D cell culture was established to provide a more physiologically relevant environment. This approach utilizes biomaterials to imitate the extracellular matrix, which helps to rejuvenate cells and restore their natural properties in physiological environments.[1] In the realm of cellular research, 3D culture systems, though superior to their 2D counterparts, fall short in emulating the intricate heterogeneity of in vivo cell growth and differentiation. In an endeavor to address this shortcoming, scientists have advocated the employment of 3D culture systems to cultivate and steer stem cells toward organoid formations, structures that bear a closer resemblance to human tissues. By providing a more physiologically relevant environment, organoids could improve our understanding of bone development, regulation, and disease mechanisms, ultimately leading to better treatments and outcomes for patients.
长期以来，骨缺损、肿瘤和其他与骨骼相关的疾病一直是主要的健康问题。与骨骼和关节损伤相关的漫长恢复时间表不仅危及患者的健康，还会造成相当大的经济影响。例如，骨关节炎是一种影响全球数百万人的疾病，有惊人的 3.03 亿人患有膝关节和髋关节骨关节炎。鉴于这些挑战，迫切需要更好地了解骨骼发育、调节、疾病机制、进展和药物筛选。目前，传统的实验室研究方法依赖于 2D 细胞培养和动物实验。然而，这些方法具有明显的局限性，包括缺乏空间结构、细胞外基质和生理条件下的细胞-环境相互作用。因此，它们不能完全模拟体内发现的复杂和异质条件。为了克服这些限制，建立了 3D 细胞培养以提供更具生理相关性的环境。这种方法利用生物材料来模拟细胞外基质，这有助于使细胞恢复活力并恢复它们在生理环境中的自然特性。1 在细胞研究领域，3D 培养系统虽然优于 2D 培养系统，但在模拟体内细胞生长和分化的复杂异质性方面存在不足。为了解决这一缺点，科学家们提倡使用 3D 培养系统来培养干细胞并将其引导至类器官形成，即与人体组织更相似的结构。 通过提供更生理相关的环境，类器官可以提高我们对骨骼发育、调节和疾病机制的理解，最终为患者带来更好的治疗和结果。
Organoids are complex structures that are created through the cultivation of pluripotent stem cells or progenitor cells derived from tissue. These cells undergo a process of differentiation and self-organization/material-induced organization, culminating in the formation of 3D cell culture systems. Within these systems, an ensemble of matrices often emerges, closely mirroring the extracellular matrix (ECM). Such foundational structures pave the way for the flourishing and maturation of organoids.[2] Organoid constructs are gaining widespread attention in the field of oncology due to their potential applications in cultivating tumor cells for drug screening and emulating the growth of tumors in vivo. Beyond their noted applications, the exploration of organoids extends into realms such as regenerative medicine.[3, 4] For example, gut organoids, with their inherent simplicity, have garnered extensive scrutiny, offering profound insights into the intricacies of intestinal evolution and restorative processes.[5] In the neural realm, brain organoids have emerged as a recent frontier. Numerous investigations delve into these structures, elucidating the underpinnings of neurodevelopmental maladies. Concurrently, a subset of these studies harnesses brain organoids as a tool for pharmacological screenings targeting cerebral ailments.[6, 7] Moreover, bone organoids have recently gained attention as they provide a sophisticated and physiologically relevant 3D culture system that mimics bone construction under physiological conditions. Bone organoids, by offering a physiologically pertinent milieu, hold the potential to enrich our comprehension of bone development, regulation, and pathological mechanisms, thereby paving the way for enhanced patient treatments and outcomes. However, while numerous 3D bone tissue engineering models have been developed, a significant portion of them comprise a singular cellular component and simplistic designs, limiting their capacity to faithfully replicate the dynamic in vitro microenvironment.[1, 8] Viewed through this lens, bone organoids emerge as exemplary frameworks, adept at facilitating the emulation of in vivo microenvironments and physiological orchestration. This provides accessible models for studies of bone development, dynamic regulation of bone, and tissue regeneration, in addition to opening new pathways for disease diagnosis and drug screening (Figure 1).[9
类器官是通过培养多能干细胞或源自组织的祖细胞而产生的复杂结构。这些细胞经历分化和自组织/材料诱导的组织过程，最终形成 3D 细胞培养系统。在这些系统中，经常会出现一组基质，与细胞外基质 （ECM） 紧密镜像。这样的基础结构为类器官的繁荣和成熟铺平了道路。2 类器官构建体在肿瘤学领域受到广泛关注，因为它们在培养肿瘤细胞以进行药物筛选和模拟体内肿瘤生长方面具有潜在应用。除了他们著名的应用之外，对类器官的探索还延伸到再生医学等领域。3、4例如，肠道类器官凭借其固有的简单性，已经获得了广泛的审查，为肠道进化和恢复过程的复杂性提供了深刻的见解。5 在神经领域，大脑类器官已成为最近的前沿领域。大量研究深入研究了这些结构，阐明了神经发育疾病的基础。同时，这些研究的一个子集利用脑类器官作为针对脑部疾病的药理学筛选工具。6、7此外，骨类器官最近受到了关注，因为它们提供了一种复杂且生理相关的 3D 培养系统，可以模拟生理条件下的骨骼结构。 骨类器官通过提供生理相关的环境，有可能丰富我们对骨骼发育、调节和病理机制的理解，从而为增强患者治疗和结果铺平道路。然而，虽然已经开发了许多 3D 骨组织工程模型，但其中很大一部分由单一的细胞成分和简单的设计组成，限制了它们忠实复制动态体外微环境的能力。1、8从这个角度来看，骨类器官成为典型框架，擅长促进体内微环境和生理编排的模拟。这为骨骼发育、骨骼动态调控和组织再生的研究提供了可用的模型，此外还为疾病诊断和药物筛选开辟了新的途径（图1）。9]
骨类器官设计和应用场景的示意图。A） 骨解剖学和组织学。B） 构建骨类器官的程序和方法。C） 骨相关细胞增殖和分化的重要信号通路。D） 骨类器官的未来应用方向。
The bone is a remarkable living organ that undergoes continuous resorption and reconstruction, and the dynamic balance between these processes is crucial to maintaining bone health and mechanical strength. This intricate procedure is underscored by a confluence of cells and molecular entities, underscoring the imperative of discerning the interplay between the skeletal architecture and the cellular microhabitat. In the intricate landscape of bone research, bone organoids have burgeoned as a captivating methodology, offering insights into bone maturation and diseases while shaping in vitro paradigms for pharmacological evaluations and toxicity probes. Beyond mere models, these organoids hold promise in repairing scaffolds, aiding the restitution of bone deficits and fostering the renaissance of injured skeletal tissue. In the present review, we overview the intricate biological signals and microenvironment during bone development, explore the latest advancements in bone organoid construction, and discuss their potential challenges and applications in the future.
骨骼是一个非凡的活器官，经历着持续的吸收和重建，这些过程之间的动态平衡对于维持骨骼健康和机械强度至关重要。细胞和分子实体的汇合强调了这一复杂的过程，强调了辨别骨骼结构和细胞微生境之间相互作用的必要性。在骨研究的复杂领域中，骨类器官已成为一种引人入胜的方法，它提供了对骨成熟和疾病的见解，同时为药理学评估和毒性探针塑造了体外范式。这些类器官不仅仅是模型，还有望修复支架，帮助恢复骨骼缺损，并促进受伤骨骼组织的复兴。在本文中，我们概述了骨骼发育过程中错综复杂的生物信号和微环境，探讨了骨类器官构建的最新进展，并讨论了它们在未来的潜在挑战和应用。
2 Physiological Microenvironment of the Bone Tissue
2 骨组织的生理微环境
Bone is organized, dynamic mineralized connective tissue, comprising not only the inorganic substance of hydroxyapatite (HAp) but also organic components such as collagen, supplementary proteins, and fats.[10, 11] HAp is crucial for bone mineralization, imparting structural stability and enabling the bone to bear weight under mechanical stress. Concurrently, other organic components ensure the bone retains its flexibility and elasticity. Bone is mainly constituted by four cells (Figure 2B): i) Osteoblasts—which comprise 4–6% of resident osteocytes and line the bone surface—are specialized cuboidal epithelial cells replete with organelles. Their principal duties encompass the synthesis of bone matrix proteins and facilitating calcification. Essentially, they underpin new bone tissue formation and its apt mineralization, pivotal for bone strength and architecture. ii) Osteocytes, the fully matured form of osteoblasts, constitute the majority (90–95%) of all bone cells and have an impressive lifespan. These cells reside within tiny cavities called lacunae that are embedded within the mineralized bone matrix, and they exhibit a distinctive dendritic sHApe. Interestingly, the strategic positioning of osteocytes is thought to play a vital role in the bone's mechanosensitive function. Essentially, the location of these cells between the bone's layers allows them to detect and respond to mechanical forces, contributing to the bone's ability to adapt and strengthen in response to physical stress. Essentially, the location of these cells between the bone's layers allows them to detect and respond to mechanical forces, contributing to the bone's ability to adapt and strengthen in response to physical stress. iii) Situated on bone surfaces, bone lining cells, a distinct subclass of osteoblasts, are flattened and quiescent. Generally, they remain peripheral to direct bone formation or resorption processes. Under specific physiological conditions, bone lining cells can activate and secrete substances, potentially contributing to bone remodeling and repair. iv) Emerging from stem cell differentiation, pre-osteoblasts represent osteoblast precursors: flattened entities devoid of bone matrix synthesis capability. v) Originating from the hematopoietic stem cell lineage, osteoclasts are mature, multinucleated entities responsible for bone resorption, and recent research indicates they also secrete cytokines pivotal for immune regulation and inflammation.[8, 10, 11] Within the orchestration of the aforementioned cells, bone perpetually rejuvenates and restructures in reaction to mechanical stress, chiefly harmonizing bone resorption with formation. Temporal regulation of this equilibrium sees osteoblast-associated entities (comprising osteoblasts, osteocytes, and bone lining cells) and resorption-linked osteoclasts collectively organized into bone multicellular units (BMUs), inclusive of their progenitors. Osteoblast-associated cells orchestrate bone matrix production and mineralization; while, osteoclast-linked cells facilitate its dissolution primarily via acidic compound generation. This remodeling paradigm, chiefly governed by humoral regulation, ensures calcium and phosphorus homeostasis within the body. Osteoblast-associated cells are responsible for the production and mineralization of bone matrix; osteoclast-associated cells dissolve and resorb bone matrix mainly through the production of acidic compounds. This remodeling process is mainly regulated by humoral regulation and is used to maintain calcium and phosphorus stability in the body.[12
骨骼是有组织的、动态的矿化结缔组织，不仅包括羟基磷灰石 （HAp） 的无机物质，还包括胶原蛋白、补充蛋白和脂肪等有机成分。10、11HAp 对骨矿化至关重要，赋予骨骼结构稳定性并使骨骼能够在机械应力下承受重量。同时，其他有机成分确保骨骼保持其柔韧性和弹性。骨骼主要由四个细胞组成（图2B）：i） 成骨细胞——占常驻骨细胞的 4-6% 并排列在骨骼表面——是充满细胞器的特殊立方体上皮细胞。它们的主要职责包括骨基质蛋白的合成和促进钙化。从本质上讲，它们支撑着新骨组织的形成及其适当的矿化作用，对骨骼强度和结构至关重要。ii） 骨细胞是完全成熟的成骨细胞形式，占所有骨细胞的大部分 （90-95%），并且具有令人印象深刻的寿命。这些细胞位于称为腔隙的微小腔内，这些腔嵌入矿化骨基质中，它们表现出独特的树突状 sHApe。有趣的是，骨细胞的战略定位被认为在骨骼的机械敏感功能中起着至关重要的作用。从本质上讲，这些细胞在骨骼层之间的位置使它们能够检测和响应机械力，从而有助于骨骼适应和加强以应对物理压力的能力。从本质上讲，这些细胞在骨骼层之间的位置使它们能够检测和响应机械力，从而有助于骨骼适应和加强以应对物理压力的能力。 iii） 位于骨表面的骨衬里细胞（成骨细胞的一个独特亚类）扁平且静止。通常，它们对直接的骨形成或吸收过程保持边缘。在特定的生理条件下，骨衬里细胞可以激活和分泌物质，可能有助于骨骼重塑和修复。iv） 从干细胞分化中出现的前成骨细胞代表成骨细胞前体：缺乏骨基质合成能力的扁平实体。v） 破骨细胞起源于造血干细胞谱系，是负责骨吸收的成熟多核实体，最近的研究表明，它们还分泌对免疫调节和炎症至关重要的细胞因子。8、10、11在上述细胞的协调中，骨骼在机械应力的作用下不断恢复活力和重组，主要是协调骨骼吸收与形成。这种平衡的时间调节将成骨细胞相关实体（包括成骨细胞、骨细胞和骨衬里细胞）和再吸收连接的破骨细胞集体组织成骨多细胞单位 （BMU），包括它们的祖细胞。成骨细胞相关细胞协调骨基质的产生和矿化;而破骨细胞连接细胞主要通过酸性化合物的产生促进其溶解。这种重塑范式主要受体液调节控制，确保体内钙和磷的稳态。成骨细胞相关细胞负责骨基质的产生和矿化;破骨细胞相关细胞主要通过产生酸性化合物来溶解和重吸收骨基质。 这种重塑过程主要受体液调节调节，用于维持体内钙和磷的稳定性。12]
骨膜和骨重塑界面微环境。A） 骨表面覆盖着骨膜，骨膜可分为内层和外层，具有相应的成分。B） 骨组织主要由四种类型的细胞组成：成骨细胞、骨细胞、骨衬里细胞和前成骨细胞。成骨细胞起源于间充质干细胞，破骨细胞起源于单核细胞-巨噬细胞。
From the macroscopic morphology, bones can generally be classified into long, short, flat bones, and irregular bones. Long bones, found in extremities, are elongated with marrow cavities housing red marrow in childhood and transitioning to yellow marrow in adulthood. Short bones, located in hands and feet, possess columnar or cubic shapes, underpinning the intricate actions of humans. Flat bones, resembling panels, are prevalent in cranial and chest walls, primarily safeguarding vital organs. Irregular bones, scattered across the body, serve diverse functions; for instance, the butterfly bone shapes the cranial cavity; while, vertebrae support and protect the spinal cord.[13
从宏观形态上看，骨骼一般可分为长骨、短骨、扁骨和不规则骨。在四肢发现的长骨被拉长，在儿童时期有红骨髓的骨髓腔，在成年后过渡到黄骨髓。位于手和脚中的短骨呈柱状或立方体形状，支撑着人类复杂的动作。扁平骨，类似于面板，普遍存在于颅壁和胸壁中，主要保护重要器官。散布在全身的不规则骨骼具有不同的功能;例如，蝴蝶骨塑造了颅腔;同时，椎骨支撑和保护脊髓。13]
Microscopically, most bones comprise two primary matrix forms: cortical and trabecular bone. Cortical bone, denser and on the bone's periphery, has a low porosity of 5–15% and robust compression resistance; while, trabecular bone is more porous at 40–90%. Cortical bone consists of organized bone lamellae, categorized into osteons, circumferential, and interstitial lamellae. Osteons or Haversian systems feature a central Haversian canal with tissue fluid, blood vessels, and nerves, encircled by 10–20 concentric lamellae. Neighboring central canals interconnect via Volkmann's canal, facilitating nutrient delivery to adjacent bone cells. The circumferential bone lamella is delineated into outer and inner domains. The outer stratum envelops the cortical bone's exterior surface with 10–40 lamellar layers, whereas the inner lamella, skirting the inner facet of the cortical bone, are comparatively slender and irregular. Spaces between adjacent osteons and circumferential lamellae are bridged by interosseous plates, ensuring cohesive cortical bone connectivity. Trabecular bone is a type of bone tissue that is mainly encompassed by the bone cortex, which is a more permeable tissue that can facilitate the development and specialization of hematopoietic cells (Figure 3). Trabeculae are the foundational units of trabecular bone, and their arrangement contrasts with the uniformity of cortical bone, often being shaped by mechanical stresses on the bone.[14, 15] Bone marrow tissue, primarily found in the cavities of long bone ends, houses diverse hematopoietic stem cells and offers an optimal microenvironment for their differentiation and growth.
在显微镜下，大多数骨骼包括两种主要基质形式：皮质骨和小梁骨。皮质骨，更致密，位于骨骼的外围，具有 5-15% 的低孔隙率和强大的抗压性;而小梁骨的孔隙率更高，为 40-90%。皮质骨由有组织的骨片组成，分为骨片、圆周骨片和间质片。骨质或 Haversian 系统的特点是中央 Haversian 管，其中包含组织液、血管和神经，周围环绕着 10-20 个同心薄片。邻近的中央管通过 Volkmann 管相互连接，促进营养物质输送到相邻的骨细胞。圆周骨片分为外部和内部区域。外层用 10-40 层片状层包裹着皮质骨的外表面，而绕过皮质骨内侧的内层则相对较细长且不规则。相邻骨细胞和周向薄片之间的空间由骨间板桥接，确保有凝聚力的皮质骨连接。骨小梁骨是一种主要由骨皮层包围的骨组织，骨皮层是一种渗透性更强的组织，可以促进造血细胞的发育和特化（图3）。小梁是骨小梁的基本单位，它们的排列与皮质骨的均匀性形成鲜明对比，皮质骨通常由骨骼上的机械应力形成。14、15 元骨髓组织主要存在于长骨末端的空腔中，容纳着多种造血干细胞，并为它们的分化和生长提供了最佳的微环境。
骨骼的基本结构。骨骼可分为皮质骨和小梁骨。皮质骨由骨质和骨片组成，小梁骨由骨小梁组成。
Besides the bone, surrounding accessory tissues include the periosteum, nerves, lymphatic, and blood vessels, with bone marrow within long bones. The periosteum, a vascular-rich connective tissue, envelopes the bone's outer surface and attaches via Sharpey fibers. Comprising a dense outer layer rich in elastic fibers and fibroblasts and an inner osteogenic layer with osteoblasts, osteoclasts, and osteoprogenitor cells, it ensures firm connectivity. Based on function, some researchers delineate the periosteum into three layers from outermost to innermost: the fibrous, undifferentiated, and cambium layers. These layers respectively serve protective, stress transfer, and osteogenic functions (Figure 2A). Intrabony nerves, predominantly found in cortical bone, cancellous bone, and periosteum, are primarily sensory and visceromotor. They detect nociception and can release neurotransmitters influencing bone metabolism. Creating and using bone organoids demands attention to the bone's complex, dynamic structure and its interactions with tissues and organs. Successfully crafting functional bone organoids necessitates understanding the intricate regulatory mechanisms of bone development and its communication with the body. Addressing these factors is vital for long-term effective organoid creation.
除骨骼外，周围的辅助组织包括骨膜、神经、淋巴管和血管，其中骨髓位于长骨内。骨膜是一种富含血管的结缔组织，包裹着骨骼的外表面并通过 Sharpey 纤维连接。它由一个富含弹性纤维和成纤维细胞的致密外层和一个带有成骨细胞、破骨细胞和成骨祖细胞的内成骨层组成，确保牢固的连接。根据功能，一些研究人员将骨膜从最外层到最内层分为三层：纤维层、未分化层和形成层。这些层分别起到保护、应力转移和成骨功能（图 2A）。骨内神经主要见于皮质骨、松质骨和骨膜，主要是感觉和内脏运动。它们检测伤害感受并可以释放影响骨代谢的神经递质。创建和使用骨类器官需要注意骨骼复杂的动态结构及其与组织和器官的相互作用。成功制作功能性骨类器官需要了解骨骼发育及其与身体通信的复杂调节机制。解决这些因素对于长期有效的类器官创建至关重要。
3 Signaling Pathways Involved in Bone Organoids
骨类器官涉及的 3 个信号通路
It is important to have a clear understanding of the cellular and molecular mechanisms involved in bone regeneration to construct bone organoids. Various signaling pathways that play crucial roles in bone development and fracture healing have been identified and studied. The core concept behind building organoids is to control how stem cells differentiate and proliferate at the early stages, which ultimately leads to the formation of organized structures resembling natural tissues. By manipulating important factors in the signaling pathways associated with bone development and repair, we can finely tune the way different types of cells differentiate and arrangement. This enables us to create flexible and precise control over the growth and distribution of various cells within the organoids. Therefore, gaining knowledge about these pathways is crucial for successful bone organoid construction (Figure 4). This section will describe several relevant and significant signaling pathways in bone development and repair.[16] Although these pathway-related studies were partly completed under in-vivo experimental conditions, it is also of exploratory and summary significance for the construction of bone organoids in vitro.
清楚地了解骨再生所涉及的细胞和分子机制以构建骨类器官非常重要。已经确定和研究了在骨骼发育和骨折愈合中起关键作用的各种信号通路。构建类器官背后的核心概念是控制干细胞在早期阶段的分化和增殖方式，最终导致形成类似于天然组织的有组织结构。通过操纵与骨骼发育和修复相关的信号通路中的重要因素，我们可以微调不同类型细胞的分化和排列方式。这使我们能够对类器官内各种细胞的生长和分布进行灵活而精确的控制。因此，获得有关这些途径的知识对于成功的骨类器官构建至关重要（图4）。本节将介绍骨骼发育和修复中的几种相关且重要的信号通路。16 尽管这些与通路相关的研究部分是在体内实验条件下完成的，但它对体外骨类器官的构建也具有探索性和总结性意义。
骨骼发育中的相关信号通路。有必要全面概述影响成骨细胞增殖和发育的重要信号通路，以更好地了解参与骨形成的分子机制。使用 BioRender.com 创建。A） MAPK 通路，B） Wnt 通路，C） TGFβ 和 BMPs 通路，D） Notch 通路，以及 E） PI3K/AKT 通路。
3.1 Wnt/β-Catenin Signaling Pathway
3.1 Wnt/β-连环蛋白信号通路
Wnt proteins are a group of secreted molecules that can bind to Frizzled (FZD) receptors, which are transmembrane proteins consisting of seven segments. When Wnt proteins bind to FZD receptors, it triggers a cascade of events that activate the β-catenin pathway downstream.[17] Wnts are actively involved in cell proliferation, cell fate choice, and apoptosis during embryonic development, and in the maintenance of adult stem cells and repair of injuries. The typical Wnt signaling downstream through β-catenin is induced to promote osteogenesis during fracture healing, which means that the Wnt signaling pathway is involved in the differentiation and functional formation of osteoblasts.[18] Research has shown that deactivating substances that inhibit the Wnt signaling pathway can lead to a significant increase in bone density. This has been observed in experiments where the expression of Dickkopfs 1 (DKK1), a protein that binds to LRP5, has been reduced, as well as in cases where mutations in the gene responsible for producing Sclerostin, a molecule that acts as a soluble Wnt inhibitor, have resulted in decreased function and subsequently, increased bone mass.[18] It was also demonstrated by knocking down the Foxf1 gene in bone marrow stem cells (BMSCs), observing bone mineralization by ALP staining and Von Kossa staining, and by measuring the expression of downstream β-catenin to prove that the Foxf1 gene affects the osteogenesis of BMSC in vitro through the wnt signaling pathway.[19] In vitro, stem cell culture can also promote stem cell osteogenic differentiation for mineralized deposition by affecting the Wnt/β-catenin pathway. For example, construction of nano-artificial bone membranes using Poly(L-lactide-co-glycolide) (PLGA)/MgO/quercetin promotes osteogenesis and angiogenesis through the Wnt/β-catenin pathway, and the osteogenic differentiation of BMSCs is regulated by the construction of artificial bone membrane to promote bone formation.[20
Wnt 蛋白是一组分泌分子，可与卷曲 （FZD） 受体结合，FZD 受体是由七个片段组成的跨膜蛋白。当 Wnt 蛋白与 FZD 受体结合时，它会触发一系列事件，激活下游的 β-catenin 通路。17 Wnts 积极参与胚胎发育过程中的细胞增殖、细胞命运选择和细胞凋亡，以及成体干细胞的维持和损伤的修复。在骨折愈合过程中，通过 β-catenin 下游的典型 Wnt 信号转导被诱导以促进成骨，这意味着 Wnt 信号通路参与成骨细胞的分化和功能形成。18 研究表明，抑制 Wnt 信号通路的失活物质可导致骨密度显着增加。在实验中观察到这一点，其中 Dickkopfs 1 （DKK1）（一种与 LRP5 结合的蛋白质）的表达降低，以及负责产生硬化蛋白（一种充当可溶性 Wnt 抑制剂的分子）的基因突变导致功能下降，随后导致骨量增加。18 还通过敲低骨髓干细胞 （BMSC） 中的 Foxf1 基因，通过 ALP 染色和 Von Kossa 染色观察骨矿化，以及通过测量下游 β-catenin 的表达来证明 Foxf1 基因通过 wnt 信号通路在体外影响 BMSC 的成骨。19 在体外，干细胞培养还可以通过影响 Wnt/β-catenin 通路来促进干细胞成骨分化，从而实现矿化沉积。 例如，使用聚（L-丙交酯-乙交酯共聚物）（PLGA）/MgO/槲皮素构建纳米人工骨膜通过 Wnt/β-catenin 途径促进成骨和血管生成，而 BMSCs 的成骨分化受人工骨膜构建促进骨形成的调节。20]
3.2 Notch Signaling Pathway
3.2 Notch 信号通路
The Notch signaling pathway is a fundamental biological mechanism that involves the interaction between specific proteins on the surface of cells, which plays a crucial role in various cellular processes such as survival, growth, differentiation, and maintenance of normal cellular functions. This pathway is highly conserved across different species and is involved in the regulation of cell fate determination and homeostasis. Notch receptors and ligands are both cell surface transmembrane proteins. Notch signaling is initiated by bypasses between signal sending cells expressing the ligand (DSL ligands, Jagged1, Jagged2, Delta1, Delta3, or Delta4) and neighboring signal receiving cells expressing the receptor (Notch1-4).[21] Notch signaling has been found to play a crucial role in the promotion of both angiogenesis and regeneration of bone tissue. When there is a disruption in the genetic signaling of Notch, it not only affects the morphology and growth of bone vessels but also leads to various negative impacts on the skeletal system, such as reduced osteogenesis, shortened long bones, chondrocyte defects, loss of bone trabeculae, and reduced bone mass.[22] Notch-2 has also been demonstrated to promote osteoclast differentiation to accelerate bone resorption.[23] A study has verified that the Notch-2/jag1 signaling pathway is involved in the differentiation of mesenchymal stem cells to osteoblasts through animal experiments.[24] Other studies produced loss-of-function mice (LOF) by deleting alleles of the NOTCH transcriptional effector RBPjκ or by ablating the Dnmt3b Gene. By executing artificial tibial fractures in loss-of-function mice (LOF) and controls, they verified that the Notch signaling pathway contributed to maintaining the number of BMSC populations and maintaining their progenitor state, as well as promoting differentiation to osteoblasts.[25, 26] It was also shown that the Notch signaling pathway could regulate the differentiation and proliferation of MSCs, promoting the proliferation but inhibiting the differentiation of stem cells.[27] The notch signaling pathway has also been used in the design of bone regeneration materials. It has been verified by animal experiments that artificially constructed mouse models of bone defects, by local implantation of hydrogels containing valproic acid, resulted in activation of Notch pathway, upregulation of Notch 1, HES1, HEY1, and JAG1 expression, and significantly improved bone defect repair compared to the control group.[28] A research team activated the Notch pathway by synthesizing porous hydrogels presenting Jagged-1 (a mimetic hexapeptide with Notch agonist activity), which enhanced mechanotransduction signaling and osteogenesis of human mesenchymal stem cells (HMSCs), and promoted HMSCs osteogenesis and bone matrix deposition.[29
Notch 信号通路是一种基本的生物机制，涉及细胞表面特定蛋白质之间的相互作用，在存活、生长、分化和维持正常细胞功能等各种细胞过程中起着至关重要的作用。该通路在不同物种中高度保守，并参与细胞命运决定和体内平衡的调节。Notch 受体和配体都是细胞表面跨膜蛋白。Notch 信号转导是通过表达配体的信号发送细胞（DSL 配体、Jagged1、Jagged2、Delta1、Delta3 或 Delta4）和表达受体的相邻信号接收细胞 （Notch1-4） 之间的旁路启动的。21 已发现 Notch 信号转导在促进血管生成和骨组织再生中起关键作用。当 Notch 的遗传信号中断时，不仅影响骨血管的形态和生长，还会对骨骼系统造成各种负面影响，如成骨减少、长骨缩短、软骨细胞缺损、骨小梁丢失和骨量减少。22 Notch-2 也被证明可以促进破骨细胞分化以加速骨吸收。23 一项研究通过动物实验证实，Notch-2/jag1 信号通路参与间充质干细胞向成骨细胞的分化。24 其他研究通过删除 NOTCH 转录效应子 RBPjκ 的等位基因或通过消融 Dnmt3b 基因产生了功能丧失小鼠 （LOF）。 通过在功能丧失小鼠 （LOF） 和对照中执行人工胫骨骨折，他们验证了 Notch 信号通路有助于维持 BMSC 种群的数量和维持其祖细胞状态，以及促进分化为成骨细胞。25 元、26 元研究还表明，Notch 信号通路可以调节 MSCs 的分化和增殖，促进干细胞的增殖但抑制干细胞的分化。27 Notch 信号通路也已用于骨再生材料的设计。动物实验证实，人工构建骨缺损小鼠模型，通过局部植入含有丙戊酸的水凝胶，导致 Notch 通路激活，Notch 1、HES1、HEY1 和 JAG1 表达上调，与对照组相比，骨缺损修复得到显著改善。28 一个研究小组通过合成呈递 Jagged-1（一种具有 Notch 激动剂活性的模拟六肽）的多孔水凝胶来激活 Notch 通路，这增强了人间充质干细胞 （HMSC） 的机械转导信号和成骨，并促进了 HMSCs 成骨和骨基质沉积。29]
3.3 BMPs/TGF-β Pathway 3.3 BMPs/TGF-β 通路
Bone morphogenetic proteins (BMPs) are a group of proteins that belong to the transforming growth factor (TGF-β) superfamily. They are known for their ability to stimulate the differentiation of undifferentiated mesenchymal stem cells into osteoblasts, which are responsible for bone formation. BMPs interact with specific receptors called serine/threonine kinases, which initiate signaling pathways through both classical (or smad-dependent) and non-classical (or smad-independent) pathways. These pathways are important for regulating a variety of cellular processes and ultimately contribute to the development and maintenance of bone tissue.[30] The BMPs pathway was demonstrated to be an essential pathway in osteogenesis by deleting Smad1 and Smad5 in chondrocytes, which led to severe dysplasia and subsequent lack of ossification.[31] Studies also proved that bone morphogenetic protein 2 (BMP2) is required for the fracture healing process by using BMP2 gene-inactivated mice, executing artificial fractures in mice and the controls, and by observing and recording the process of fracture healing in mice, as well as proving the function of BMP2 in activating bone progenitor cells within the periosteum.[32] This pathway has been used in bone regeneration. In a study, silk fibroin hydrogels containing chitosan nanoparticles were constructed, and the excellent cartilage repair ability of this material was verified in vitro by loading TGF-β1 and BMP-2.[33
骨形态发生蛋白 （BMP） 是一组属于转化生长因子 （TGF-β） 超家族的蛋白质。它们以其刺激未分化的间充质干细胞分化为成骨细胞的能力而闻名，成骨细胞负责骨形成。BMP 与称为丝氨酸/苏氨酸激酶的特异性受体相互作用，这些受体通过经典（或 smad 依赖性）和非经典（或 smad 非依赖性）途径启动信号通路。这些通路对于调节各种细胞过程很重要，并最终有助于骨组织的发育和维持。30 BMPs 通路通过删除软骨细胞中的 Smad1 和 Smad5 被证明是成骨的重要途径，这导致严重的异型增生和随后的骨化缺失。31 研究还通过使用 BMP2 基因灭活的小鼠，在小鼠和对照中执行人工骨折，以及通过观察和记录小鼠骨折愈合过程，以及证明 BMP2 在激活骨膜内骨祖细胞中的功能，证明骨形态发生蛋白 2 （BMP2） 是骨折愈合过程所必需的。32 该途径已用于骨再生。在一项研究中，构建了含有壳聚糖纳米颗粒的丝素蛋白水凝胶，并通过加载 TGF-β1 和 BMP-2 在体外验证了该材料优异的软骨修复能力。33]
3.4 MAPK Pathway 3.4 MAPK 通路
Mitogen-activated protein kinases (MAPKs) are an ancient group of serine/threonine kinases that mediate responses to a wide range of stimuli.[34] There are four main branching pathways of the MAPK pathway: ERK, JNK, p38/MAPK, and ERK5. There was a study observing bone development and bone density changes in mice by knocking out the mouse p38α gene, the upstream regulation of the MAPK pathway, proving the important role of MAPK for bone development and preventing bone resorption.[35] It was also demonstrated that Fgf9 inhibits osteogenesis by downregulating the MAPK pathway, mice with knockout of Fgf9 showed increased bone mass, and that these changes were realized through the MAPK pathway.[36] Some studies also indicated the involvement of MAPK in osteo-inductive biomaterial-mediated gene regulation. A study investigated the mechanism of BCP ceramics in osteo-conduction. Inhibition of ERK and p38 by using specific inhibitors significantly reduced the level of osteogenesis induced by BCP ceramics. Compared to controls, the use of BCP ceramics was found to increase the expression of various markers that are specific to the development of bone tissue, such as Runx2, OSX, COL-1, ALP, BSP, and OCN. However, the positive effects of BCP ceramics on gene expression were reduced when inhibitors were used to block the ERK1/2 and p38 signaling pathways. The expression of genes related to bone development was significantly decreased in these conditions.[37] This study demonstrated the positive effects of using zinc silicate/nano-hydroxyapatite/collagen (ZS/HA/Col) scaffolds on bone regeneration and angiogenesis, while also exploring the underlying mechanisms. The researchers found that the ZS/HA/Col scaffolds created an optimal environment for bone formation and encouraged the differentiation of monocytes into TRAP+ cells, which were capable of secreting pro-osteogenic cytokines. Further, the study examined the role of MAPK in the osteogenesis process by inhibiting p38. After p38 inhibition, the number of TRAP+ cells and the secretion of cytokines (SDF-1, TGFβ1, and PDGF-BB) were significantly reduced and the bone regeneration capacity was decreased.[38
丝裂原活化蛋白激酶 （MAPK） 是一组古老的丝氨酸/苏氨酸激酶，可介导对多种刺激的反应。34 MAPK 通路有四个主要分支通路：ERK、JNK、p38/MAPK 和 ERK5。有一项研究通过敲除小鼠 p38α 基因（MAPK 通路的上游调节）来观察小鼠的骨骼发育和骨密度变化，证明了 MAPK 对骨骼发育和防止骨吸收的重要作用。35 还证明 Fgf9 通过下调 MAPK 通路来抑制成骨，敲除 Fgf9 的小鼠显示骨量增加，并且这些变化是通过 MAPK 通路实现的。36 一些研究还表明 MAPK 参与骨诱导生物材料介导的基因调控。一项研究调查了 BCP 陶瓷在骨传导中的机制。使用特异性抑制剂抑制 ERK 和 p38 显着降低了 BCP 陶瓷诱导的成骨水平。与对照组相比，发现使用 BCP 陶瓷会增加对骨组织发育具有特异性的各种标志物的表达，例如 Runx2、OSX、COL-1、ALP、BSP 和 OCN。然而，当使用抑制剂阻断 ERK1/2 和 p38 信号通路时，BCP 陶瓷对基因表达的积极影响降低。在这些情况下，与骨骼发育相关的基因的表达显著降低。37 这项研究证明了使用硅酸锌/纳米羟基磷灰石/胶原蛋白 （ZS/HA/Col） 支架对骨再生和血管生成的积极影响，同时也探索了潜在的机制。 研究人员发现，ZS/HA/Col 支架为骨形成创造了最佳环境，并促进单核细胞分化为 TRAP+ 细胞，这些细胞能够分泌促成骨细胞因子。此外，该研究通过抑制 p38 检查了 MAPK 在成骨过程中的作用。p38 抑制后，TRAP+ 细胞数量和细胞因子 （SDF-1 、 TGFβ1 和 PDGF-BB） 分泌显著降低，骨再生能力降低。38 元]
3.5 PI3K/Akt Pathway 3.5 PI3K/Akt 通路
Many cytokines, hormones, and so on can initiate the PI3K/Akt pathway by activating receptor tyrosine kinases (RTK) and G protein-coupled receptors (GPCRs), which subsequently activate PI3K to produce phospholipids to activate downstream effectors. PI3K/AKT plays a role in many cellular processes, including cell cycle, cell survival, inflammation, metabolism, and apoptosis.[39] It was demonstrated that miRNA-21 can promote the osteogenesis process of MSCs through upregulation of PTEN/PI3K/Akt/HIF-1α pathway. Implanting Lenti-miRNA-21/β-TCP/BMSC scaffolds in a rat model of cranial defects accelerates the healing of cranial defects.[40] There is a study demonstrating that microRNA-29b-3p, encapsulated in extracellular vesicles derived from bone marrow MSCs, can promote fracture healing by inhibiting PTEN gene expression and activating the PIK3/Akt pathway.[41
许多细胞因子、激素等可以通过激活受体酪氨酸激酶 （RTK） 和 G 蛋白偶联受体 （GPCR） 来启动 PI3K/Akt 通路，随后激活 PI3K 产生磷脂以激活下游效应子。PI3K/AKT 在许多细胞过程中发挥作用，包括细胞周期、细胞存活、炎症、代谢和细胞凋亡。39 研究表明，miRNA-21 可以通过上调 PTEN/PI3K/Akt/HIF-1α 通路促进 MSCs 的成骨过程。在大鼠颅骨缺损模型中植入 Lenti-miRNA-21/β-TCP/BMSC 支架可加速颅骨缺损的愈合。40 有一项研究表明，封装在骨髓 MSC 来源的细胞外囊泡中的 microRNA-29b-3p 可以通过抑制 PTEN 基因表达和激活 PIK3/Akt 通路来促进骨折愈合。41]
3.6 IGF Pathway 3.6 IGF 通路
Insulin-like growth factors (IGFs) are crucial players in various biological processes, such as promoting cell growth and preventing cell death, as well as supporting normal growth and development. Among the members of the IGF family, IGF1 and IGF2 are the only two. IGF has several functions, including promoting the differentiation of osteoblasts, enhancing the deposition of bone matrix, and promoting the expression of collagen and other non-collagenous proteins in the body.[42] It was demonstrated by experimental studies using IGF-1 knockout mutant mice to evaluate the effect of IGF-1 on bone development, and IGF-1 knockout mice were demonstrated to have decreased bone density and trabecular density in all bones of the body.[43] By knocking out the IGF-1 receptor in mouse pre-osteoblasts, one study found that both osteogenesis and mineralization processes were affected in the knocked-out cells.[44] According to a recent study, the use of BMP-6 in combination with IGF-1 could potentially be a more effective option than BMP-2 when it comes to promoting enhanced bone formation and mineralization in both orthopedic surgery and bone tissue engineering applications.[45] One study developed a dual delivery system that accelerated the healing of critical-sized cranial defects in rats by releasing (BMP2) and insulin-like growth factor 1 (IGF1) sequentially in microparticles (MPs), along with an injectable alginate/collagen (Alg/Col)-based hydrogel. One of the important pathways promoting bone healing is activation through IGF-1.[46
胰岛素样生长因子 （IGF） 在各种生物过程中发挥着关键作用，例如促进细胞生长和防止细胞死亡，以及支持正常生长和发育。在 IGF 家族的成员中，IGF1 和 IGF2 是仅有的两个。IGF 具有多种功能，包括促进成骨细胞的分化、增强骨基质的沉积以及促进体内胶原蛋白和其他非胶原蛋白的表达。42 通过使用 IGF-1 敲除突变小鼠评估 IGF-1 对骨骼发育的影响的实验研究表明，IGF-1 敲除小鼠在身体所有骨骼中的骨密度和小梁密度降低。43 通过敲除小鼠前成骨细胞中的 IGF-1 受体，一项研究发现敲除细胞中的成骨和矿化过程都受到影响。44 根据最近的一项研究，在骨科手术和骨组织工程应用中，在促进增强骨形成和矿化方面，BMP-6 与 IGF-1 联合使用可能是比 BMP-2 更有效的选择。45 一项研究开发了一种双重递送系统，该系统通过在微粒 （MP） 中依次释放 （BMP2） 和胰岛素样生长因子 1 （IGF1） 以及基于可注射藻酸盐/胶原蛋白 （Alg/Col） 的水凝胶来加速大鼠临界大小的颅骨缺损的愈合。促进骨愈合的重要途径之一是通过 IGF-1 激活。46]
3.7 FGF Pathway 3.7 FGF 通路
Fibroblast growth factor (FGF) plays a crucial role in the regulation of cell proliferation and differentiation during bone development, specifically for osteoblasts and fibroblasts. Moreover, FGF is involved in the process of bone resorption and osteogenesis, as well as in other essential cellular processes such as angiogenesis and wound healing. Its impact on these processes highlights the significant influence FGF has on the overall growth and development of bone tissue.[47] Twenty-three members of the fibroblast growth factors (FGFs) family have been identified, which is a large family of peptides.[48] A study that cultured rat bone marrow cells in FGF-2-containing medium in vitro revealed a significant increase in cell size and cell number, as well as a significant increase in BMP and osteopontin expression.[49] However, it was also demonstrated that overexpression of FGF-2 inhibits osteoblast differentiation and bone formation.[50] Integrating the above perspectives, a study investigated the function of the material in slow release of FGF-2 in vivo and the upper dose of FGF-2 to promote the osteogenesis by constructing FGF-2 knockout mice and using Col/HA scaffolds loaded with FGF-2.[51] A novel material loaded with nWH/nBG and FGF-18 via chitin-PLGA hydrogel had been developed. By comparing the novel material with the currently commonly used commercial bioceramic (HAP) loaded FGF-18, it demonstrated a higher degree of recovery in mice cranial defects and found that FGF-18 is non-replaceable by other members of the FGF family in this combination.[52] There was also some research to design basic fibroblast growth factor (bFGF)-loaded mesoporous silica nanoparticles (MSN) nanocomposites to demonstrate the effect of FGF-2 on osteoblast differentiation by in vitro experiments, to analyze the downstream activated genes, and to verify that the nanocomposites can promote the repair of bone defects by in vivo experiments in mice.[53
成纤维细胞生长因子 （FGF） 在骨骼发育过程中细胞增殖和分化的调节中起着至关重要的作用，特别是对于成骨细胞和成纤维细胞。此外，FGF 参与骨吸收和成骨过程，以及其他重要的细胞过程，如血管生成和伤口愈合。它对这些过程的影响凸显了 FGF 对骨组织整体生长和发育的重大影响。47 已鉴定出成纤维细胞生长因子 （FGF） 家族的 23 个成员，这是一个大的肽家族。48 一项在含 FGF-2 的体外培养基中培养大鼠骨髓细胞的研究显示，细胞大小和细胞数量显著增加，BMP 和骨桥蛋白表达显著增加。49 然而，研究也证明 FGF-2 的过表达会抑制成骨细胞分化和骨形成。50 整合上述观点，一项研究通过构建 FGF-2 敲除小鼠和使用负载 FGF-2 的 Col/HA 支架，调查了该材料在体内 FGF-2 缓释和 FGF-2 上限剂量促进成骨的功能。51 已经开发了一种通过几丁质-PLGA 水凝胶加载 nWH/nBG 和 FGF-18 的新型材料。通过将这种新材料与目前常用的商用生物陶瓷 （HAP） 负载的 FGF-18 进行比较，它在小鼠颅骨缺损中显示出更高的恢复度，并发现 FGF-18 在这种组合中不能被 FGF 家族的其他成员取代。52 还有一些研究设计了载有碱性成纤维细胞生长因子 （bFGF） 的介孔二氧化硅纳米颗粒 （MSN） 纳米复合材料，以通过体外实验证明 FGF-2 对成骨细胞分化的影响，分析下游激活基因，并通过小鼠体内实验验证纳米复合材料可以促进骨缺损的修复。53 元]
Although the various pathways involved in the growth and development of bone have been studied independently, they are intricately linked and operate in different spatial and temporal contexts. While each pathway has distinct upstream and downstream components, they also interact with each other. To create bone organoids, it is crucial to replicate the developmental and growth processes of natural bone tissue. Understanding the molecular biology behind bone growth and development is essential in developing in vitro models that can mimic these processes and create complex, functional, and bionic bone organoids. Moreover, comprehending these biological mechanisms is vital in devising effective strategies for constructing and applying bone organoids. We can control when and where stem cells turn into specialized cells by using biomaterials and signaling molecules that work like traffic signals in the body's pathways.
尽管已经独立研究了骨骼生长和发育所涉及的各种途径，但它们错综复杂地联系在一起，并在不同的空间和时间背景下运作。虽然每条通路都有不同的上游和下游成分，但它们也会相互相互作用。为了创建骨类器官，复制天然骨组织的发育和生长过程至关重要。了解骨骼生长和发育背后的分子生物学对于开发可以模拟这些过程并创建复杂、功能性和仿生骨类器官的体外模型至关重要。此外，理解这些生物机制对于设计构建和应用骨类器官的有效策略至关重要。我们可以通过使用生物材料和信号分子来控制干细胞何时何地变成专门的细胞，这些分子的作用就像身体通路中的交通信号一样。
4 Component Elements of Bone Organoids
骨类器官的 4 个组成部分
The development of bone organoids is still in its early stages, but there are a few key components that are crucial to the process. These include finding the right type of cells to use, as well as using bioactive materials that can mimic the extracellular matrix. While there are some techniques for constructing organoids that don't require scaffolding materials, many of the current strategies still rely on bioactive materials. In order to successfully create bone organoids, it is important to carefully consider the specific types of cells and materials being used in the construction process (Figure 5).
骨类器官的开发仍处于早期阶段，但有一些关键组成部分对该过程至关重要。这些包括寻找合适的细胞类型来使用，以及使用可以模拟细胞外基质的生物活性材料。虽然有一些不需要支架材料的构建类器官的技术，但目前的许多策略仍然依赖于生物活性材料。为了成功创建骨类器官，重要的是要仔细考虑构建过程中使用的特定类型的细胞和材料（图5）。
骨类器官的构建方法。骨类器官的细胞来源于临床获得的骨组织碎片或干细胞，包括 MSC、ESC、hiPSC 和 hPDC。模拟细胞基质的生物材料包括天然聚合物材料和合成高分子材料。支架是通过 3D 打印、静电纺丝和其他策略构建的。使用 BioRender.com 创建。A） 细胞来源。B） 生物材料。C） 建设策略。
4.1 Cell Origins for Bone Organoids
4.1 骨类器官的细胞起源
Cells can be derived from the primary tissue or differentiated from pluripotent stem cells.[54] As described in Part 1, the development of bone involves a diversity of cell types, including osteogenesis-associated cells, bone resorption-associated cells, and hematopoietic- associated cells.
细胞可以来源于原代组织或从多能干细胞分化而来。54 如第 1 部分所述，骨骼的发育涉及多种细胞类型，包括成骨相关细胞、骨吸收相关细胞和造血相关细胞。
Osteogenesis-associated cells mainly include osteoblasts and stem cells that can differentiate into osteoblasts. A study was conducted by constructing 3D porous collagen–hydroxyapatite scaffolds and culturing human primary osteoblasts (obtained through Promocell) to demonstrate the potential of this scaffold in bone tissue engineering.[55] An in vitro co-culture system of osteoblasts and osteocytes based purely on human primary cells had also been established, and osteocytes and preosteoblasts were obtained by isolation from the femoral head of hip replacement patients.[56] The advantages of primary cells derived from primary tissues are that cells are more similar to physiological conditions, retaining many important markers and functions in vivo and maintaining heterogeneity. The disadvantages; however, are that the osteoblasts themselves have a weak proliferative capacity and cannot be used on a large scale.
成骨相关细胞主要包括成骨细胞和可分化成骨细胞的干细胞。通过构建 3D 多孔胶原蛋白-羟基磷灰石支架和培养人类原代成骨细胞（通过 Promocell 获得）进行了一项研究，以证明该支架在骨组织工程中的潜力。55 还建立了完全基于人原代细胞的成骨细胞和骨细胞体外共培养系统，通过从髋关节置换患者的股骨头分离获得骨细胞和成骨细胞前体。56 来源于原代组织的原代细胞的优点是细胞与生理条件更相似，在体内保留了许多重要的标志物和功能，并保持了异质性。缺点;然而，成骨细胞本身的增殖能力较弱，不能大规模使用。
Attempts to construct bone organoids from pluripotent stem cells have also been made, including human induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), human-periosteum-derived cells (hPDCs), and embryonic stem cells (hESCs).
还尝试从多能干细胞构建骨类器官，包括人诱导多能干细胞 （iPSC）、间充质干细胞 （MSC）、人骨膜衍生细胞 （hPDC） 和胚胎干细胞 （hESC）。
iPSs refer to somatic cells that have been genetically “reprogrammed” back to an embryonic stem cell state. In 2016, one study discovered that iPSCs cultured in osteogenic medium could successfully obtain osteoblast-like cells and that adding resveratrol supported the osteogenic differentiation of iPSCs. Dexamethasone-induced apoptosis of osteoblast-like cells was effectively inhibited by pretreatment with resveratrol.[57] It was demonstrated that retinoic acid (RA) can induce osteogenic differentiation of human induced pluripotent stem cells (hiPSCs) to osteoblast-like and osteoclast-like cells, and it was found that the induction of bone formation is dependent on cellular signaling of RA receptors RARα and RARβ and activates BMP (bone morphogenetic protein) and Wnt signaling pathways.[58] Through this discovery, a team designed 3D printed Ti6Al4V (3DTi) scaffolds loaded with iPSCs and induced differentiation by retinoic acid(RA) to accelerate the repair of mandibular defects in mice.[59] A recent study utilized a 3D rotational suspension culture system to induce the differentiation of induced pluripotent stem cells (iPSCs) toward articular cartilage regeneration or vascularized bone regeneration via endochondral osteogenesis. This was achieved by regulating key signaling pathways, namely BMP-4 and FGF-2, through mechanical stimulation.[60] These studies demonstrate the significant application of iPSC in osteogenesis. However, the challenges to be tackled regarding the utilization of iPS as a cell source are the instability of differentiation, including subsequent tumorigenesis and teratoma formation. Besides this, other challenges include being time-consuming, poor reproducibility, low efficiency, and low survival rates of transplanted cells.
iPS 是指在基因上被“重新编程”回胚胎干细胞状态的体细胞。2016 年，一项研究发现，在成骨培养基中培养的 iPSC 可以成功获得成骨细胞样细胞，并且添加白藜芦醇支持 iPSC 的成骨分化。地塞米松诱导的成骨细胞样细胞凋亡通过白藜芦醇预处理得到有效抑制。57 证明视黄酸 （RA） 可以诱导人诱导的多能干细胞 （hiPSC） 成骨分化为成骨细胞样和破骨细胞样细胞，并且发现骨形成的诱导依赖于 RA 受体 RARα 和 RARβ 的细胞信号传导，并激活 BMP（骨形态发生蛋白）和 Wnt 信号通路。58 通过这一发现，一个团队设计了 3D 打印的 Ti6Al4V （3DTi） 支架，该支架加载了 iPSC，并通过视黄酸 （RA） 诱导分化，以加速小鼠下颌骨缺损的修复。59 最近的一项研究利用 3D 旋转悬浮培养系统诱导多能干细胞 （iPSC） 通过软骨内成骨向关节软骨再生或血管化骨再生分化。这是通过机械刺激调节关键信号通路，即 BMP-4 和 FGF-2 来实现的。60 这些研究表明 iPSC 在成骨中的重要应用。然而，关于利用 iPS 作为细胞来源需要解决的挑战是分化的不稳定性，包括随后的肿瘤发生和畸胎瘤形成。 除此之外，其他挑战包括耗时、可重复性差、效率低和移植细胞存活率低。
MSCs can be classified as umbilical cord-derived MSCs (UC-MSCs), adipose tissue-derived MSCs (AT-MSCs), and bone marrow MSCs (BMSCs). Although they all belong to MSCs, there remains a lot of variability due to their origin. UC-MSCs exhibit a faster proliferation rate and can be maintained for a longer number of passages, but their isolation efficiency is lower. The advantages of AT-MSCs are their convenience of isolation and their higher proliferation rate and higher colony formation capacity than BMSCs.[61] In contrast, BMSCs benefit from their immune evasion properties and higher application value, in addition to the fact that BMSCs tend to differentiate more toward bone and cartilage than the other two types of MSCs. Bone marrow MSCs play a crucial role in bone formation and have been a research hotspot in recent years.[62] A study established a mouse model of femur fracture and demonstrated that systemic and local application of bone marrow mesenchymal stem cells can promote fracture healing through direct differentiation into osteoblasts.[63] It was demonstrated that bone marrow mesenchymal stem cells transplanted via tail vein could be mobilized by erythropoietin to the bone defect area and involved in the regeneration of new bone, obtaining better osteogenic and angiogenic therapeutic results.[64] A study was conducted where researchers aimed to improve the ability of MSCs to promote bone regeneration. To achieve this, they designed 3D sponge-like scaffolds with hierarchical and interconnected pores using a low-temperature deposition modeling (LDM) printing technique. These scaffolds were successful in enhancing the paracrine functions of MSCs, resulting in effective bone regeneration.[65] It has also been investigated to reprogram the function and fate of MSCs by designing customized microcryogels with dual functional units to construct osteochondral organoid.[66] It appeared to be found that the proliferation and differentiation of MSCs could be better regulated by 3D modeling, leading to applications in organoid construction.
MSC 可分为脐带衍生的 MSC （UC-MSC）、脂肪组织衍生的 MSC （AT-MSC） 和骨髓 MSC （BMSC）。尽管它们都属于 MSCs，但由于它们的来源仍然存在很多可变性。UC-MSC 表现出更快的增殖速率，并且可以维持更长的传代次数，但其分离效率较低。AT-MSC 的优势在于分离方便，比 BMSC 具有更高的增殖率和更高的集落形成能力61。相比之下，BMSCs 受益于其免疫逃避特性和更高的应用价值，此外，BMSCs 比其他两种类型的 MSC 更倾向于向骨骼和软骨分化。骨髓 MSC 在骨形成中起着至关重要的作用，近年来一直是研究热点年。62 一项研究建立了股骨骨折的小鼠模型，并证明骨髓间充质干细胞的全身和局部应用可以通过直接分化成骨细胞来促进骨折愈合。63 研究表明，通过尾静脉移植的骨髓间充质干细胞可以被促红细胞生成素动员到骨缺损区域并参与新骨的再生，获得更好的成骨和血管生成治疗效果。64 进行了一项研究，研究人员旨在提高 MSC 促进骨再生的能力。为了实现这一目标，他们使用低温沉积建模 （LDM） 打印技术设计了具有分层和互连孔的 3D 海绵状支架。 这些支架成功地增强了 MSC 的旁分泌功能，从而实现了有效的骨再生。65 还研究了通过设计具有双功能单元的定制微冷冻凝胶来构建骨软骨类器官来重新编程 MSC 的功能和命运。66 似乎发现 MSC 的增殖和分化可以通过 3D 建模更好地调节，从而在类器官构建中得到应用。
Similar to mesenchymal stem cells, human-periosteum-derived cells (hPDCs) are a class of cells that originate from the periosteum with osteogenic capacity. The important function of periosteum in bone regeneration process brought hPDCs much attention in bone organoids construction. A study assembled organoid by serum-free pretreatment of human periosteal-derived cells, which were simultaneously initiated with bone morphogenetic protein 2 (BMP-2). By generating a more efficient progenitor cell population, the process of cartilage osteogenesis was mimicked in vivo in mice.[67] Other studies revealed possible cell–material interactions between CaP and hPDCs by combining in vitro pretreated hPDCs with CaP-based biomaterial carriers and explored their possible gene regulation.[68] The hPDCs were proliferated in vitro under serum-free conditions and seeded as seed cells to form micro-organoid in 3D printed thiolene alginate hydrogels. The micro-organoids were induced into chondrogenic and osteogenic differentiation under the cytokines supplied and constituted 3D callus organoids.[69] These studies may enable hPDCs to become key cells in the construction of bone organoids.
与间充质干细胞类似，人骨膜衍生细胞 （hPDC） 是一类起源于骨膜的具有成骨能力的细胞。骨膜在骨再生过程中的重要功能使 hPDCs 在骨类器官构建中受到广泛关注。一项研究通过对人骨膜衍生细胞进行无血清预处理来组装类器官，这些细胞同时由骨形态发生蛋白 2 （BMP-2） 启动。通过产生更有效的祖细胞群，在小鼠体内模拟了软骨成骨的过程。67 其他研究通过将体外预处理的 hPDC 与基于 CaP 的生物材料载体相结合，揭示了 CaP 和 hPDCs 之间可能的细胞-材料相互作用，并探索了它们可能的基因调控。68 hPDCs 在无血清条件下体外增殖，并作为种子细胞接种，在 3D 打印的硫代烯海藻酸盐水凝胶中形成微类器官。微类器官在细胞因子供应下被诱导进行软骨形成和成骨分化，并构成 3D 愈伤组织类器官。69 这些研究可能使 hPDC 成为构建骨类器官的关键细胞。
ESCs are important multipotent stem cell types with the ability to self-renew infinitely in vitro and to differentiate into all cell lineages of the tricellular lineage. However, hESC research is a very controversial topic because the process of ESC isolation involves embryo destruction; in addition, ESC from nuclear transfer may be used for reproductive cloning. A study implanted mouse ESCs on ceramic scaffolds and demonstrated that mouse ESCs can upregulate osteogenic markers such as Cbfa-1/Runx2, osteopontin, bone sialoprotein, and osteocalcin to promote differentiation toward cartilage and ultimately osteogenesis; the scaffold can be used to modulate the spatial and temporal differentiation of ESCs.[70] Many studies are currently replacing human embryonic stem cells with induced stem cells.
ESC 是重要的多能干细胞类型，能够在体外无限自我更新并分化为三细胞谱系的所有细胞谱系。然而，hESC 研究是一个非常有争议的话题，因为 ESC 分离的过程涉及胚胎破坏;此外，来自核转移的 ESC 可用于生殖克隆。一项研究将小鼠 ESC 植入陶瓷支架上，并证明小鼠 ESC 可以上调成骨标志物，如 Cbfa-1/Runx2、骨桥蛋白、骨唾液蛋白和骨钙素，以促进向软骨分化并最终成骨;支架可用于调节 ESC 的空间和时间分化。70 目前许多研究正在用诱导干细胞取代人类胚胎干细胞。
Bone resorption-associated cells are osteoclasts and cells that can differentiate into osteoclast-associated cells. Osteoclasts originate from hematopoietic monocytes-macrophages, which are highly differentiated multinucleated giant cells. Osteogenesis and bone resorption counterbalance each other in a physiological environment, maintaining a dynamic equilibrium to remodel the mechanical properties of bone. M-CSF and RANKL are the main factors driving osteoclast proliferation and differentiation. Currently, the construction of bone organoids is mostly based on osteogenic-associated cells, and the balance between osteogenic-associated cells and osteoblast-associated cells may be considered in the construction of delicate organoids in the future.
骨吸收相关细胞是破骨细胞，可以分化为破骨细胞相关细胞。破骨细胞来源于造血单核细胞-巨噬细胞，它们是高度分化的多核巨细胞。成骨和骨吸收在生理环境中相互制衡，保持动态平衡以重塑骨骼的机械性能。M-CSF 和 RANKL 是驱动破骨细胞增殖和分化的主要因素。目前，骨类器官的构建主要基于成骨相关细胞，未来构建精细类器官时可能会考虑成骨相关细胞和成骨细胞相关细胞之间的平衡。
Hematopoietic-associated cells mainly consist of hematopoietic cells and their progenitor cells. These cells in the bone marrow play a key role in connective tissue regeneration and blood vessel formation. A study was conducted to generate mesenchymal, vascular, and bone marrow hematopoietic components by using a hybrid matrix hydrogel containing Matrigel and collagen types I and IV to form a 3D structure similar to the bone marrow niche.[71] Other studies have used polyethylene glycol (PEG) and hyaluronic acid (HA) to mimic the bone marrow niche; cultured hBMSC, hematopoietic cells and progenitor cells (hSPC), and bone marrow related stomal cells; and formed well-constructed bone marrow organoids under the regulation of BMP-2.[72
造血相关细胞主要由造血细胞及其祖细胞组成。骨髓中的这些细胞在结缔组织再生和血管形成中起关键作用。进行了一项研究，通过使用含有基质胶和 I 型和 IV 型胶原蛋白的杂交基质水凝胶来生成间充质、血管和骨髓造血成分，以形成类似于骨髓生态位的 3D 结构。71 其他研究使用聚乙二醇 （PEG） 和透明质酸 （HA） 来模拟骨髓生态位;培养的 hBMSC、造血细胞和祖细胞 （hSPC） 以及骨髓相关造孔细胞;并在 BMP-2 的调节下形成结构良好的骨髓类器官。72]
In addition to using the above stem cell differentiation alone, some studies have been completed by combining two or even more cells for the construction of biomimetic bone models. The advantage of this construction is that it allows easier control of the types of cells in different regions, but the disadvantage lies in the finer control required for the regulation of the different cells. One study constructed a composite implant in vitro using carboxymethyl chitosan, alginate, bone marrow mesenchymal stem cells, and endothelial progenitor cells, which was then transplanted into the femoral head of rabbits after core decompression surgery to treat steroid-induced ONFH. The implant composite promoted bone and blood vessel growth and facilitated disease repair.[73] This strategy of using multiple cells and its regulation may also be an important approach for the future in vitro construction of bone organoids.
除了单独使用上述干细胞分化外，还通过结合两个甚至多个细胞构建仿生骨骼模型完成了一些研究。这种结构的优点是它更容易控制不同区域的细胞类型，但缺点在于调节不同细胞所需的更精细的控制。一项研究使用羧甲基壳聚糖、藻酸盐、骨髓间充质干细胞和内皮祖细胞在体外构建了复合植入物，然后在核心减压手术后将其移植到兔的股骨头，以治疗类固醇诱导的 ONFH。植入物复合材料促进骨骼和血管生长并促进疾病修复。73 这种使用多个细胞的策略及其调节也可能是未来体外构建骨类器官的重要方法。
4.2 Biomaterials Mimicking ECM
4.2 模拟 ECM 的生物材料
The extracellular matrix (ECM) provides the environment in which cells survive in vivo, and each of the different tissue cells survive in a diverse matrix. It mechanically and biochemically directs the behaviors of cells, playing an essential role for maintaining tissue functions and health. The ECM influences cell fates via its specific physical properties (e.g., matrix stiffness, geometry, matrix porosity, fiber diameter, and so on) and cell–cell interactions (cell junctions, cytokines, etc.).[74] Compared with natural matrices, the construction of organoid requires precise design of the relevant ECMs in vitro, and many biomaterials are currently used to reproduce the properties of natural matrices for the construction of organoids. The bone ECM contains organic and inorganic components with little water content and is composed primarily of type I collagen (> 95% dry weight) and a small amount of amorphous matrix. Its structure undergoes a transformation from woven bone to lamellar bone.
细胞外基质 （ECM） 提供了细胞在体内生存的环境，并且每种不同的组织细胞都在不同的基质中生存。它以机械和生化方式指导细胞的行为，在维持组织功能和健康方面发挥着重要作用。ECM 通过其特定的物理特性（例如，基质刚度、几何形状、基质孔隙率、纤维直径等）和细胞间相互作用（细胞连接、细胞因子等）影响细胞命运。74 与天然基质相比，类器官的构建需要在体外精确设计相关的 ECM，目前许多生物材料用于复制天然基质的特性以构建类器官。骨 ECM 包含含水量很少的有机和无机成分，主要由 I 型胶原蛋白 （> 95% 干重） 和少量无定形基质组成。它的结构经历了从编织骨到层状骨的转变。
The Engelbreth–Holm–Swarm (EHS) matrix is a significant component in the advancement of the organoid field. This matrix is a reconstituted basement membrane that is obtained from mouse sarcoma and is popularly known by the trade names Matrigel, Geltrex, and Cultrex BME. Its essential role in providing a suitable extracellular matrix for cell growth and differentiation has been instrumental in the success of organoid cultures.[2] However, the high cost of EHS matrix materials and the unclear chemical properties, as well as the variation among different products leading to bad reproducibility, lead to its limited application in organoid construction.
Engelbreth-Holm-Swarm （EHS） 矩阵是类器官领域发展的重要组成部分。该基质是从小鼠肉瘤中获得的重组基底膜，商品名为 Matrigel、Geltrex 和 Cultrex BME。它在为细胞生长和分化提供合适的细胞外基质方面发挥着重要作用，有助于类器官培养的成功。2 然而，EHS 基质材料的高成本和不明确的化学性质，以及不同产品之间的差异导致重现性差，导致其在类器官构建中的应用受到限制。
The earliest biomaterial applied in bone organoids design was the demineralized bone matrix (DBM), formed primarily by decalcification of fresh allogeneic or xenogeneic bone; and therefore, prone to rejection reactions. The naturally available bone matrix components and cytokines provide good biological properties, osteo-conductivity, and biodegradation, resulting in an excellent material for promoting osteogenesis and bone healing.[75] However, the uncontrolled heterogeneity of the material composition prevents them from quality assessment and accelerated osteogenesis mechanisms. In addition, the use of DBM alone does not provide a 3D scaffold structure. However, there is still much research in combining it with other biomaterials because of its practicality and economy. One study designed a new method of fabricating DBM and investigated its gene expression that promoted osteogenesis and demonstrated a promising effect in critically sized bone regeneration.[76] There are also studies combining DBM with other scaffold materials. One study demonstrated the outstanding ability to promote osteogenesis by constructing a polycaprolactone (PCL)/β-tricalcium phosphate (β-TCP) scaffold and loading nano-DBM onto the surface of the PCL/β-TCP composite scaffold using a lyophilization method.[77
最早应用于骨类器官设计的生物材料是脱矿质骨基质 （DBM），主要由新鲜同种异体或异种体骨脱钙形成;因此，容易出现排斥反应。天然存在的骨基质成分和细胞因子具有良好的生物学特性、骨传导性和生物降解性，是促进成骨和骨愈合的极好材料。75 然而，材料成分不受控制的异质性使它们无法进行质量评估和加速成骨机制。此外，单独使用 DBM 并不能提供 3D 支架结构。然而，由于其实用性和经济性，仍有许多研究将其与其他生物材料相结合。一项研究设计了一种制造 DBM 的新方法，并研究了其促进成骨的基因表达，并证明了在临界大小的骨再生中具有有希望的效果。76 还有一些研究将 DBM 与其他支架材料相结合。一项研究表明，通过构建聚己内酯 （PCL）/β-磷酸三钙 （β-TCP） 支架并使用冻干方法将纳米 DBM 加载到 PCL/β-TCP 复合支架表面，具有促进成骨的出色能力。77]
Bioactive materials for bone organoids design can be classified as inorganic bone materials and organic materials. Hydroxyapatite (HAp) is one of the most commonly applied inorganic biocompatible materials. This is because HAp is the most predominant inorganic component of bone, with excellent osteo-conduction and osteo-induction properties, and is also less prone to inflammatory reactions in the body.[78] In addition, the modern technology enables easy control of the crystallinity, porosity, and morphology of HAp. In recent years, the HAp material scaffold has been capable of modulating its porosity and alignment. A study has constructed a biomimetic hierarchical scaffold by using surface minerization in combination with 3D bio-mapping techniques, which facilitates the adhesion and differentiation of MSCs and also improves capillary regeneration after implantation.[79] However, due to the drawbacks of the HAp, many current strategies for constructing scaffolds combine the application of HAp with other materials. One study constructed MgAlEu-layered double hydroxide (MAE-LDH) nanosheets on porous HAp scaffolds, which increased bone regeneration and angiogenesis by activating the Wnt/β-catenin signaling pathway.[80] Other studies have combined organic materials, such as the construction of silk fibroin (SF)/hydroxyapatite (HAp) scaffolds embedded with naringin polylactic acid-glycolic acid (PLGA) microspheres, to validate their excellent bone regeneration capacity by inoculation with bone mesenchymal stem cells.[81] Along with HAp, tricalcium phosphate is one of the most commonly used inorganic materials. In contrast to HAp, β-TCP is resorbable; it can be dissolved and absorbed by osteoclasts; and thus, easily replaced by new bone.[82] A recent study utilized low-temperature rapid prototyping (LT-RP) technology to create a novel porous scaffold made of magnesium powder, PLGA, and β-tricalcium phosphate (β-TCP), known as PLGA/TCP/magnesium (PTM). The findings revealed that the PTM scaffold demonstrated a remarkable ability to improve new bone formation and enhance the mechanical properties of the newly formed bone.[83] BMP-2 releasing gelatin/β-TCP sponges have also been constructed and have been shown to promote bone regeneration by histological and computed tomography examination.[84
用于骨类器官设计的生物活性材料可分为无机骨材料和有机材料。羟基磷灰石 （HAp） 是最常用的无机生物相容性材料之一。这是因为 HAp 是骨骼中最主要的无机成分，具有优异的骨传导和骨诱导特性，并且在体内也不易发生炎症反应。78 此外，现代技术可以轻松控制 HAp 的结晶度、孔隙率和形态。近年来，HAp 材料支架已经能够调节其孔隙率和对准性。一项研究通过使用表面矿物化结合 3D 生物映射技术构建了一个仿生分层支架，这促进了 MSC 的粘附和分化，也改善了植入后的毛细血管再生。79 然而，由于 HAp 的缺点，目前许多构建支架的策略都将 HAp 的应用与其他材料相结合。一项研究在多孔 HAp 支架上构建了 MgAlEu 层状双氢氧化物 （MAE-LDH） 纳米片，通过激活 Wnt/β-catenin 信号通路来增加骨再生和血管生成。80 其他研究结合了有机材料，例如构建嵌入柚皮苷聚乳酸-乙醇酸 （PLGA） 微球的丝素蛋白 （SF）/羟基磷灰石 （HAp） 支架，以验证它们通过接种骨间充质干细胞来验证其优异的骨再生能力。81 与 HAp 一样，磷酸三钙是最常用的无机材料之一。 与 HAp 相比，β-TCP 是可吸收的;可被破骨细胞溶解和吸收;因此，很容易被 New Bone 取代。82 最近的一项研究利用低温快速原型制作 （LT-RP） 技术创造了一种由镁粉、PLGA 和β-磷酸三钙 （β-TCP） 制成的新型多孔支架，称为 PLGA/TCP/镁 （PTM）。研究结果显示，PTM 支架显示出改善新骨形成和增强新形成骨的机械性能的显着能力。还构建了 83 个 BMP-2 释放明胶/β-TCP 的海绵，并通过组织学和计算机断层扫描检查显示可促进骨再生。84 元]
Organic bioactive materials used for bone organoids design include natural polymeric materials such as silk fibroin (SF), Gelatin, chitosan, and HA, and synthetic polymeric materials such as PCL, PLA, and PEG. In recent years, many extracellular matrix mimicking strategies utilize more than single biomaterial.[85] The combination of the materials together may lead to better properties and more versatile combinations. One study designed elastic-like peptides (ELPs)-modified SF to provide 3D adhesion sites for cells to promote BMSc adhesion, proliferation, and differentiation in vitro; while, demonstrating the excellent bone regeneration ability through animal experiments.[86] A ternary PCL/SF/MgZnSiO4 scaffold was designed to mimic the unique mechanical properties and fiber diameter of the bone matrix, and the ability of this scaffold to promote mineralization and induce osteogenic differentiation of stem cells was demonstrated by in vitro experiments.[87] In a recent study, researchers developed scaffolds for bone regeneration using a combination of gelatin, alginate, and cerium oxide nanoparticles. These scaffolds were fabricated using a freeze-drying method and showed improved mechanical properties, as well as the ability to scavenge reactive oxygen species (ROS) and promote biomineralization. The researchers also observed that the addition of cerium oxide to the scaffold enhanced stem cell osteogenic differentiation, indicating its potential for use in bone organoid construction.[88] A study fabricated biocompatible polyamide-6/chitosan fiber scaffolds by electrostatic spinning technology and verified their excellent osteogenic ability with the ability to promote osteoblast attachment and proliferation.[89] Another study fabricated PVA/CS/ HAp nanofiber composites by electrostatic spinning, which enhanced the mechanical properties of the scaffolds and verified that they provided attachment points for osteoblasts and promoted the ossification process.[90] A study has also mimicked the bone marrow niche using PEG and Gelatine, which mimics the morphology and function of bone marrow by implanting a variety of stem cells.[72, 91
用于骨类器官设计的有机生物活性材料包括天然聚合物材料，如丝素蛋白 （SF）、明胶、壳聚糖和 HA，以及合成聚合物材料，如 PCL、PLA 和 PEG。近年来，许多细胞外基质模拟策略使用的不仅仅是单一的生物材料。85 这些材料的组合可能会带来更好的性能和更通用的组合。一项研究设计了弹性样肽 （ELP） 修饰的 SF，为细胞提供 3D 粘附位点，以促进 BMSc 在体外的粘附、增殖和分化;同时，通过动物实验展示了优异的骨再生能力。86 三元 PCL/SF/MgZnSiO4 支架被设计为模拟骨基质独特的机械性能和纤维直径，体外实验证明了该支架促进干细胞矿化和诱导成骨分化的能力。87 在最近的一项研究中，研究人员使用明胶、藻酸盐和氧化铈纳米颗粒的组合开发了用于骨再生的支架。这些支架是使用冷冻干燥方法制造的，显示出改进的机械性能，以及清除活性氧 （ROS） 和促进生物矿化的能力。研究人员还观察到，在支架中添加氧化铈增强了干细胞成骨分化，表明其在骨类器官构建中的潜力。88 一项研究通过静电纺丝技术制造了生物相容性聚酰胺-6/壳聚糖纤维支架，并验证了它们优异的成骨能力以及促进成骨细胞附着和增殖的能力。89 另一项研究通过静电纺丝制备了 PVA/CS/HAp 纳米纤维复合材料，增强了支架的力学性能，并验证了它们为成骨细胞提供附着点并促进了骨化过程。90 一项研究还使用 PEG 和明胶模拟了骨髓生态位，明胶通过植入多种干细胞来模拟骨髓的形态和功能。72、91 元]
The development of biomaterials that can imitate the extracellular matrix for the purpose of constructing organoids is still in its early stages, and it poses several challenges that need to be addressed. One of the major challenges is that different types of cells have unique demands on the extracellular matrix, which means that significant adjustments to the material are required when replacing seed cells. Finding suitable materials that can meet these demands can be a labor-intensive process. Another challenge is that the structure of bones is highly complex, with different regions having different mechanical properties. For instance, the mechanical properties of cortical bone and bone marrow are significantly different, which places high demands on the materials used. Therefore, developing materials that can meet these different physical properties while also being biocompatible is crucial. Lastly, there is currently no standardized method for producing biocompatible materials in large quantities.
可以模仿细胞外基质以构建类器官的生物材料的开发仍处于早期阶段，它提出了一些需要解决的挑战。主要挑战之一是不同类型的细胞对细胞外基质有独特的要求，这意味着在更换种子细胞时需要对材料进行重大调整。寻找能够满足这些需求的合适材料可能是一个劳动密集型过程。另一个挑战是骨骼的结构非常复杂，不同的区域具有不同的机械性能。例如，皮质骨和骨髓的机械性能差异很大，这对所使用的材料提出了很高的要求。因此，开发既能满足这些不同物理特性又具有生物相容性的材料至关重要。最后，目前还没有大批量生产生物相容性材料的标准化方法。
In the development of bone organoids, it is imperative for researchers to not only replicate the macroscopic morphological features of tissues and organs using biomaterials but also to meticulously emulate the micromechanical properties inherent in the extracellular matrix of these organoids. This strategic approach is essential to furnish a conducive milieu for the growth and developmental mimicry of stem cells.
在骨类器官的开发中，研究人员不仅要使用生物材料复制组织和器官的宏观形态特征，而且还要仔细模拟这些类器官细胞外基质中固有的微观力学特性。这种战略方法对于为干细胞的生长和发育模拟提供有利的环境至关重要。
The contemporary emphasis on biomaterials emulating the extracellular matrix for organoid construction principally encompasses considerations of stiffness, viscoelasticity, degradability, and cell–matrix interaction (Table 1). Stiffness, denoting the resistance of an object to deformation under applied stress, is integral to this paradigm. Notably, anisotropy characterizes the physiological configuration of bone, manifesting as directional variance in stiffness under disparate stress orientations. A comprehensive literature survey pertaining to diverse bone mechanics has facilitated the categorization of stiffness levels, with a discernible sequential decrement observed from cortical bone to trabecular bone, cartilage, and bone marrow. Concurrently, the exploration of viscoelastic properties, encompassing storage modulus and loss modulus, has garnered attention. Given the inherent methodological dissimilarities in sampling and testing approaches, direct parameter comparisons prove impractical; thus, the scrutiny of viscoelasticity predominantly hinges on the observation of stress relaxation phenomena in cortical bone, trabecular bone, cartilage, and bone marrow. The protracted temporal dynamics of bone repair and regeneration underscore the significance of material degradation timelines, prompting a meticulous consideration of such temporal parameters in biomaterial design. Moreover, the intricate interplay between matrix materials and cellular interactions, pivotal to organoid construction, remains a focal point. In this nascent theoretical juncture, emphasis is accorded to the extensively investigated RGD sequences, which exhibit specific binding affinity to integrins. As expounded in Part I, the nuanced constituents of bone warrant a meticulous examination, and the ensuing table elucidates pertinent mechanical properties of cortical bone, trabecular bone, cartilage, and bone marrow in a compartmentalized manner.
当代对模拟细胞外基质用于类器官构建的生物材料的强调主要包括对刚度、粘弹性、可降解性和细胞-基质相互作用的考虑（表1）。刚度表示物体在施加应力下对变形的抵抗力，是这种范式不可或缺的一部分。值得注意的是，各向异性表征了骨骼的生理构型，表现为在不同应力方向下刚度的方向变化。一项与不同骨力学有关的综合文献调查促进了硬度水平的分类，从皮质骨到小梁骨、软骨和骨髓观察到明显的连续下降。同时，对粘弹性的探索，包括储能模量和损耗模量，也引起了人们的关注。鉴于采样和测试方法固有的方法差异，直接参数比较被证明是不切实际的;因此，对粘弹性的审查主要取决于对皮质骨、小梁骨、软骨和骨髓中应力松弛现象的观察。骨骼修复和再生的旷日持久的时间动态强调了材料降解时间表的重要性，促使在生物材料设计中仔细考虑这些时间参数。此外，基质材料和细胞相互作用之间错综复杂的相互作用对类器官构建至关重要，仍然是一个焦点。在这个新兴的理论节点中，重点放在广泛研究的 RGD 序列上，这些序列表现出对整合素的特异性结合亲和力。 正如第一部分所阐述的，骨骼的细微成分值得仔细检查，下表以分区的方式阐明了皮质骨、小梁骨、软骨和骨髓的相关机械特性。
表 1. 骨骼成分的相关材料特性。
| Types 类型 | Stiffness 刚度 | Viscoelasticity (stress relaxation time) 粘弹性（应力松弛时间） |
Degradability requests 可降解性请求 | Cell–matrix interactions 细胞-基质相互作用 | References 引用 |
|---|---|---|---|---|---|
| Cortical bone 皮质骨 | 10–30 GPa 10–30 加仑 | Percentage of remaining stress after 500 s is ≈95% 500 秒后剩余应力的百分比为 ≈95% |
>6 months >6 个月 |
RGDS, GRGDS, RGDS、GRGDS、 RGDfk, RGDfk， YRGDS YLGDS |
[15, 128-131] [15， 128-131] |
| Trabecular bone 骨小梁 | 0.8–15 GPa 0.8–15 加仑 | Percentage of remaining stress after 300 s is ≈90% 300 秒后剩余应力的百分比为 ≈90% |
>10 weeks >10 周 | [15, 132-135] [15， 132-135] |
|
| Cartilage 软骨 | 0.5–10 Mpa 0.5–10 米帕 | Stress reduced to 50% of initial stress is ≈0.5–10 s 应力降低到初始应力的 50% 为 ≈0.5–10 s |
>4 weeks >4 周 |
RGDfk, YRGDS, RGDfk、YRGDS、 RGDS, GCGYGRGDSPG RGDS、GCGYGRGDSPG |
[131, 136-139] [131， 136-139] |
| Bone marrow 骨髓 | 0.25–24.7 kPa 0.25–24.7 千帕 | Stress reduced to 50% of initial stress is ≈10 s 应力降低到初始应力的 50% 为 ≈10 s |
>12 days >12 天 | Not mentioned 未提及 | [140, 141] [140， 141] |
4.3 Attempted Construction of Bone Organoids
4.3 尝试构建骨类器官
Different types of bone cells are present in a special extracellular matrix, which is a set of networks composed of collagen and minerals in constant state of change. There are two modes of physiological osteogenesis, including intramembranous osteogenesis and endochondral osteogenesis. The design of in vitro bone organoids can design the hierarchy and dynamic changes of materials by mimicking both modes and control the proliferation and differentiation of stem cells from different spatial-temporal features according to the physiological development of bone. Some attempts to construct bone organoids are listed below (Figure 6 and Table 2):
不同类型的骨细胞存在于特殊的细胞外基质中，该基质是一组由胶原蛋白和矿物质组成的网络，处于不断变化的状态。生理成骨有两种模式，包括膜内成骨和软骨内成骨。体外骨类器官的设计可以通过模拟两种模式来设计材料的层次结构和动态变化，并根据骨骼的生理发育从不同的时空特征控制干细胞的增殖和分化。下面列出了一些构建骨类器官的尝试（图6 和表2）：
构建骨类器官的当前策略。A） 在小鼠大骨缺损的情况下，BMSC 包裹的骨愈伤组织类器官在 4 周内促进骨原位快速再生。经许可转载。127 版权所有 2022，爱思唯尔。B） 由结合 DBP 表面的调节分子的定义时空图谱创建的小梁骨类器官促进了 DBP 和 TCP 上的矿物沉积。经许可转载。98 版权所有 2021，美国科学促进会。C） hBMSC 分化为成骨细胞和骨细胞的功能性 3D 自组织共培养物，形成编织骨类器官。经许可转载。94 版权所有 2021，Wiley。D） 应用 hiPSC 以类似于骨髓生成骨髓的细胞、分子和空间结构的形式生成间充质元件、骨髓细胞和“弦状”脉管系统。通过体外诱导，hiPS 成功分化为骨髓的关键成分并构建骨髓类器官。经许可转载。71 版权所有 2023，Khan， Abdullah O.， Rodriguez-Romera， Antonio，美国癌症研究协会出版。
表 2. 不同类型骨类器官的组成元素。
| Types 类型 | Cell 细胞 | Materials 材料 | Assembly 集会 | Applications 应用 | References 引用 |
|---|---|---|---|---|---|
| Callus organoids 愈伤组织类器官 | hPDCs hPDC | — | Self-assembly in custom-made mold 在定制模具中自行组装 |
Critical size defects healing of long bone 长骨的临界尺寸缺损愈合 |
[92] |
| Callus organoids 愈伤组织类器官 | hBMSCs | GelMA 凝胶MA | DLP 3D printing DLP 3D 打印 | Rapid bone repair 快速骨骼修复 | [93] |
| Callus organoids 愈伤组织类器官 | hPDCs hPDC | Thiolene alginate hydrogels 硫代烯海藻酸盐水凝胶 |
3D printing 3D 打印 | Complex bone defects healing 复杂骨缺损愈合 |
[69] |
| Woven bone organoids 编织骨类器官 | hBMSCs | Silk fibroin 丝素蛋白 | Induction of cell differentiation within hydrogels 诱导水凝胶内的细胞分化 |
Study of genetic bone-related diseases 遗传性骨相关疾病的研究 |
[94] |
| Cartilaginous organoids | hPSCs(ESCs and iPSCs) | — | Self-assembly | Healing of large bone defects | [96] |
| Cartilaginous organoids | hMSCs | GelMA | Microfluidic and 3D printing | Disease modeling and drug testing | [97] |
| Cartilaginous organoids | Chondrocytes | Agarose hydrogel | Microfluidic | Disease modeling | [142] |
| Cartilaginous organoids | Adipose-derived stem cells | Collagen hydrogel | Microfluidic | Bone defects repair | [143] |
| Woven bone organoids | hES-MPs | PolyHIPE scaffold | Microfluidic | Drug testing | [95] |
| Trabecular bone organoids | Osteoblasts | Demineralized bone matrix | Induction of cell differentiation within DBM | Study the regulation of bone remodeling | [98] |
| Trabecular bone organoids | Human osteoblasts | Demineralized bone matrix and matrigel | Induction of cell differentiation within DBM | Study the effects of microgravity and degeneration | [99] |
| Bone marrow organoids | iPSCs | Type I and IV collagens | Induction of cell differentiation within hydrogels | High-throughput drug screening | [71] |
4.3.1 Callus Organoids 4.3.1 愈伤组织类器官
During endochondral osteogenesis, MSCs actively accumulate and differentiate at the defect site to form a cartilaginous core known as the “osteo-callus,” which is followed by hypertrophy, calcification, and apoptosis, as well as subsequent vascularization and recruitment and differentiation of osteogenic progenitor cells. Callus organoids refers to newborn osteoid tissue that is produced during fracture healing/long bone development and can persist in later osteogenesis. A study based on the self-assembly of hPDCs as a developmental bioengineering strategy: self-assembly of hPDCs allowed scalable production of semi-autonomous callus organoids forming bone micro-organs at implantation, and gene expression analysis revealed that in vitro mature callus organoids are associated with gene expression patterns encountered during embryonic bone development and fracture healing. Analysis of successfully healed long bones revealed that within the time of natural healing of long bones, the bridging site produced a similar structure to the native long bones.[92] A study revealed that BMSCs encapsulated callus organoids were produced by DLP-based bioprinting technology and induced into callus organoids in chondrogenic medium in vitro. Gene expression analysis revealed that callus organoids could highly recapitulate the composition and behavior of different mesenchymal stem cells during endochondral ossification in vitro and could also achieve rapid osteogenesis in vivo after in situ implantation.[93] This class of organoid was relatively simple to construct, mostly through a single stem cell to form a callus organoid, which mostly played a role in promoting the healing of bone defects. Given the pivotal functional role ascribed to callus organoids, their geometric attributes assumed paramount significance. Consequently, primary methodologies employed in the construction of callus organoids encompassed 3D printing, notable for its capacity to precisely confer requisite geometrical configurations. Concurrently, self-organization emerges as an alternative strategy, characterized by its simplicity and cost-effectiveness. The juxtaposition of these methodologies underscores their respective advantages.
在软骨内成骨过程中，MSC 在缺损部位积极积累和分化，形成称为“骨愈伤组织”的软骨核心，随后是肥大、钙化和细胞凋亡，以及随后的血管化和成骨祖细胞的募集和分化。愈伤组织类器官是指在骨折愈合/长骨发育过程中产生的新生类骨质组织，可以在以后的成骨过程中持续存在。一项基于 hPDC 自组装作为发育生物工程策略的研究：hPDC 的自组装允许在植入时形成骨微器官的半自主愈伤组织类器官的可扩展生产，基因表达分析显示，体外成熟愈伤组织类器官与胚胎骨发育和骨折愈合过程中遇到的基因表达模式相关。对成功愈合的长骨的分析表明，在长骨自然愈合的时间内，桥接部位产生了与天然长骨相似的结构。92 一项研究表明，BMSCs 封装的愈伤组织类器官是通过基于 DLP 的生物打印技术产生的，并在体外软骨形成培养基中诱导成愈伤组织类器官。基因表达分析显示，愈伤组织类器官在体外软骨内骨化过程中可以高度概括不同间充质干细胞的组成和行为，也可以在原位植入后实现体内快速成骨。93 这类器官的构建相对简单，主要通过单个干细胞形成愈伤组织类器官，主要在促进骨缺损的愈合中发挥作用。 鉴于愈伤组织类器官的关键功能作用，它们的几何属性具有至关重要的意义。因此，构建愈伤组织类器官的主要方法包括 3D 打印，其以精确赋予必要几何配置的能力而著称。同时，自组织作为一种替代策略出现，其特点是简单性和成本效益。这些方法的并置强调了它们各自的优势。
4.3.2 Woven Bone Organoids
4.3.2 编织骨类器官
Woven bone is an embryonic and fracture-repairing structural form of bone, which is mainly characterized by an irregularly interwoven arrangement of collagen fibers, with subsequent gradual remodeling into lamellar bone. In a study, primary hBMSCs were implanted onto 3D fibroin scaffolds and cultured in osteogenic differentiation medium within a spinner-flask bioreactor with continuous stirring, allowing the cells to be exposed to mechanical stimulation through shear stress generated by fluid flow. Later studies demonstrated the development of bone organoids through self-assembled co-cultures of osteoblasts and osteocytes, which served as a functional model for woven bone formation in its early stage. However, the collagen matrix in these organoids appeared disorganized and the involvement of osteoblasts in the osteogenic remodeling process was lacking.[94] There have also been studies using polyHIPE via a microfluidic approach to support the proliferation, differentiation, and production of extracellular matrix by mesenchymal progenitor cells (hES-MPs) over a longer period of 21 days, leading to the formation of woven bone that could be used for subsequent drug screening.[95] Although woven bone is an early structure in bone development and healing, this also provides us with ideas for constructing mature bone organoids in vitro. In the context of woven bone organoids fabrication, the protracted duration associated with bone mineralization and formation necessitated the employment of a supportive matrix conducive to sustained cell growth and proliferation. Presently, hydrogels emerge as the prevailing matrix material in use for this purpose. The construction of woven bone organoids using hydrogels was typically realized through microfluidic methodologies or 3D printing. Microfluidics affords advantages for disease modeling and drug testing in terms of automated, high-throughput organoid production, enabling scalability and larger quantities compared to the relatively constrained output associated with 3D printing.
编织骨是骨骼的一种胚胎和骨折修复结构形式，其主要特征是胶原纤维不规则交织排列，随后逐渐重塑为层状骨。在一项研究中，将原代 hBMSCs 植入 3D 丝素蛋白支架上，并在旋转瓶生物反应器内的成骨分化培养基中培养，并持续搅拌，使细胞能够通过流体流动产生的剪切应力受到机械刺激。后来的研究表明，骨类器官是通过成骨细胞和骨细胞的自组装共培养而发育的，这在早期阶段是编织骨形成的功能模型。然而，这些类器官中的胶原蛋白基质似乎杂乱无章，并且缺乏成骨细胞参与成骨重塑过程。94 还有研究通过微流控方法使用 polyHIPE 来支持间充质祖细胞 （hES-MP） 在 21 天的较长时间内增殖、分化和产生细胞外基质，从而形成可用于后续药物筛选的编织骨。95 虽然编织骨是骨骼发育和愈合的早期结构，但这也为我们在体外构建成熟的骨类器官提供了思路。在编织骨类器官制造的背景下，与骨矿化和形成相关的长时间需要采用有利于持续细胞生长和增殖的支持基质。目前，水凝胶成为用于此目的的主要基质材料。 使用水凝胶构建编织骨类器官通常是通过微流体方法或 3D 打印实现的。微流体技术在自动化、高通量类器官生产方面为疾病建模和药物测试提供了优势，与与 3D 打印相关的相对受限的输出相比，可实现可扩展性和更大的数量。
4.3.3 Cartilaginous Organoids
4.3.3 软骨类器官
Cartilage osteogenesis is also one of the important ways of bone formation. A study was conducted to form callus organoids by inducing differentiation of pluripotent stem cells toward the mesodermal lineage and self-organizing without scaffolds, subsequent differentiation toward chondrocytes to become cartilaginous organoids.[96] It has also been shown that the use of gelatin-based hyaluronic acid (HA) and hydroxyapatite (HYP) induces chondrogenic and osteogenic differentiation of MSCs, as well as the ability to self-assemble into osteochondral organoids, which can be used for the repair of osteochondral interfaces.[66] Cartilaginous organoids undergo osteogenic induction, which is also a way to form bone organoids, and such construction may be able to achieve a complex interface model containing bone and cartilage through the spatial distribution of scaffolds and osteogenic induction factors. Studies have also been conducted to design hMSCs-derived miniature joint-like organoids via a microfluidic system, in which engineered osteochondral complexes, synovial-like fibrous tissues, and adipose tissues are integrated into a microfluidic bioreactor. Communication between the different tissues is facilitated while maintaining their respective phenotypes.[97] In the assembly of cartilaginous organoids, the array of methods available for selection is notably diverse, affording flexibility contingent upon specific utility requirements. Microfluidics stands out as the predominant methodology employed in the construction of cartilaginous organoids. This prevalence can be attributed to the primary applications of cartilaginous organoids, predominantly geared toward disease modeling and pharmaceutical testing. In these contexts, the emphasis shifts from intricate geometric considerations to a priority on higher yield.
软骨成骨也是骨形成的重要途径之一。进行了一项研究，通过诱导多能干细胞向中胚层谱系分化并在没有支架的情况下自组织，随后向软骨细胞分化成为软骨类器官，从而形成愈伤组织类器官。96 研究还表明，使用基于明胶的透明质酸 （HA） 和羟基磷灰石 （HYP） 可诱导 MSC 的成软骨和成骨分化，以及自组装成骨软骨类器官的能力，可用于修复骨软骨接口。66 软骨类器官经历成骨诱导，这也是形成骨类器官的一种方式，这样的构建可能能够通过支架和成骨诱导因子的空间分布实现包含骨骼和软骨的复杂界面模型。还进行了研究，通过微流控系统设计 hMSC 衍生的微型关节状类器官，其中工程化的骨软骨复合体、滑膜样纤维组织和脂肪组织被整合到微流控生物反应器中。在保持其各自表型的同时促进不同组织之间的通讯。97 在软骨类器官的组装中，可供选择的方法非常多样化，根据特定的实用要求提供灵活性。微流体技术是构建软骨类器官的主要方法。 这种普遍性可归因于软骨类器官的主要应用，主要用于疾病建模和药物测试。在这些情况下，重点从复杂的几何考虑转移到优先考虑更高的产量。
4.3.4 Trabecular Bone Organoids
4.3.4 骨小梁类器官
Trabecular bone is one of the major formations of bone. One study replicated the unmineralized bone matrix, osteopontin, with demineralized cortical bone flakes and named this biomaterial as demineralized bone paper (DBP). By co-culturing osteoblasts, osteoclasts, bone lining cells and BMMs on DBP, the 3D mechanism of trabecular bone was recapitulated, and the resorption and remodeling of trabecular bone in the physiological state, as well as the model of hematopoietic stem cell hematopoiesis, were mimicked. The regulation of cell dynamics was achieved, providing an outstanding strategy for the construction of future bone organoids.[98] The most significant problem with this model is that osteocytes, which make up more than 95% of bone cells, are not included. However, this study demonstrates that we can make trabecular bone production as an important component of bone through changes in the ECM. Another study involved seeding osteoblasts extracted from human donors onto pre-treated demineralized bone, thereby promoting their differentiation and proliferation. Subsequently, the resulting small trabecular bone fragments were implanted into Matrigel gel and further cultivated, ultimately yielding larger-sized trabecular bone organoids.[99] Owing to the stringent demands imposed by the formation of trabecular bone organoids on the extracellular matrix, prevailing investigations predominantly employ demineralized bone matrix and Matrigel gel. These naturally derived materials are selected for their commendable attributes in terms of biocompatibility and fidelity to the primitive state of organs. Despite their elevated costs and inherent heterogeneity, these materials obviate the necessity for additional methodologies, enabling the self-organization requisite for the construction of organoids that exhibit both well-defined morphology and functional characteristics.
小梁骨是骨骼的主要形态之一。一项研究用脱矿质的皮质骨片复制了未矿化的骨基质骨桥蛋白，并将这种生物材料命名为脱矿质骨纸 （DBP）。通过在 DBP 上共培养成骨细胞、破骨细胞、骨衬里细胞和 BMMs，概括了骨小梁的 3D 机制，模拟了生理状态下骨小梁的吸收和重塑，以及造血干细胞造血的模型。实现了细胞动力学的调节，为构建未来的骨类器官提供了出色的策略。98 这个模型最显着的问题是不包括占骨细胞 95% 以上的骨细胞。然而，这项研究表明，我们可以通过 ECM 的变化使小梁骨的产生成为骨骼的重要组成部分。另一项研究涉及将从人类供体中提取的成骨细胞接种到预先处理的脱矿质骨上，从而促进它们的分化和增殖。随后，将所得的小骨小梁碎片植入 Matrigel 凝胶中并进一步培养，最终产生更大尺寸的骨小梁类器官。99 由于小梁骨类器官在细胞外基质上形成的严格要求，流行的研究主要采用脱矿质骨基质和 Matrigel 凝胶。这些天然来源的材料因其在生物相容性和对器官原始状态的保真度方面的值得称道的属性而被选中。 尽管成本较高且固有的异质性，但这些材料消除了额外方法的必要性，从而能够构建表现出明确形态和功能特征的类器官所需的自组织。
4.3.5 Bone Marrow Organoids
4.3.5 骨髓类器官
Bone marrow organoids are mostly used for exploration and research of hematopoietic related diseases. One study constructed 3D structures resembling vascularized human bone marrow cavities by regulating the proliferation and differentiation of iPSCs in mixed-matrix hydrogels containing Matrigel and type I and IV collagens, providing an optimal platform for modeling and drug screening of bone marrow diseases and hematological disorders.[71
骨髓类器官多用于造血相关疾病的探索和研究。一项研究通过调节含有基质胶和 I 型和 IV 型胶原蛋白的混合基质水凝胶中 iPSCs 的增殖和分化，构建了类似于血管化人骨髓腔的 3D 结构，为骨髓疾病和血液病的建模和药物筛选提供了最佳平台。71]
5 Applications of Bone Organoids
骨类器官的 5 个应用
5.1 Developing Bone Disease Models
5.1 开发骨病模型
The establishment of bone organoids enables easy and accurate availability of required disease models. Compared to animal models, organoid disease models can eliminate species differences, more realistically mimic the human pathological microenvironment, and also be more beneficial to elucidate disease mechanisms through models.[100
骨类器官的建立可以轻松准确地获得所需的疾病模型。与动物模型相比，类器官疾病模型可以消除物种差异，更真实地模拟人类病理微环境，也更有利于通过模型阐明疾病机制。100]
5.1.1 Osteoporosis Model 5.1.1 骨质疏松症模型
Osteoporosis is a condition that affects the entire skeletal system, causing a reduction in bone density and quality, and leading to the deterioration of bone microstructure. This process weakens the bones, making them more prone to fractures. Osteoporosis can have various causes and results in increased bone fragility, which can have a significant impact on a person's overall health and quality of life.[101] Animal models of osteoporosis are generally produced by surgical removal of gonads, drug application (e.g., glucocorticoids), and gene editing, which have high costs, long cycle and many uncontrollable elements. Instead, in bone organ models, the purpose of modeling can be easily accomplished by adding cytokines that regulate osteogenesis and promote the process of osteolysis or inhibit osteogenesis, causing the rate of bone resorption to be faster than the rate of osteogenesis.
骨质疏松症是一种影响整个骨骼系统的疾病，导致骨密度和质量降低，并导致骨骼微观结构恶化。这个过程会削弱骨骼，使它们更容易骨折。骨质疏松症可能有多种原因，并导致骨骼脆性增加，这会对一个人的整体健康和生活质量产生重大影响。101 骨质疏松症的动物模型一般是通过手术切除性腺、应用药物（如糖皮质激素）和基因编辑产生的，成本高、周期长、不可控因素多。相反，在骨器官模型中，通过添加调节成骨并促进成骨过程或抑制成骨的细胞因子，使骨吸收速度快于成骨速度，可以很容易地实现建模的目的。
5.1.2 Bone Cancer Model 5.1.2 骨癌模型
Bone cancers refer to primary or secondary tumors occurring in the bone or their accessory tissues. Current bone tumor models are established mostly by collecting and transplanting clinical tumor tissues into culture media or animals. Owing to the low incidence of primary tumors in bone and the fact that bone is one of the most frequently metastasized organs, most of the current research has focused on metastatic tumors of bone. A study has demonstrated that the bone microenvironment promotes further metastasis and establishes multi-organ secondary metastasis of breast and prostate cancer cells through an epigenetic programming, suggesting the significance of constructing bone organoids for the studies of tumors.[102] A study designed a 3D model by CT scanning the microstructure at the trabecular bone of the femoral epiphysis and constructing a 3D scaffold using isoproterenol. Mesenchymal stem cells were cultured in the scaffold to promote differentiation to osteoblasts, producing a microenvironment similar to the trabecular bone, and breast cancer cells were dispersed into the model to construct a 3D model of breast cancer bone metastasis.[103] Using organoids cultures of tumor cells enables tumor cells to grow in a 3D space mimicking the human microenvironment, which is more conducive to subsequent research and screening of therapeutic approaches.
骨癌是指发生在骨骼或其辅助组织中的原发性或继发性肿瘤。目前的骨肿瘤模型主要是通过将临床肿瘤组织收集并移植到培养基或动物中来建立的。由于骨中原发性肿瘤的发生率低，并且骨骼是最常转移的器官之一，因此目前的大部分研究都集中在骨转移性肿瘤上。一项研究表明，骨微环境通过表观遗传编程促进乳腺癌和前列腺癌细胞的进一步转移并建立多器官继发性转移，表明构建骨类器官对肿瘤研究的重要性。102 一项研究通过对股骨骺小梁骨的微观结构进行 CT 扫描并使用异丙肾上腺素构建 3D 支架，设计了一个 3D 模型。在支架中培养间充质干细胞，促进分化为成骨细胞，产生类似于骨小梁的微环境，将乳腺癌细胞分散到模型中，构建乳腺癌骨转移的 3D 模型。103 使用肿瘤细胞的类器官培养使肿瘤细胞能够在模拟人类微环境的 3D 空间中生长，这更有利于后续研究和治疗方法的筛选。
5.1.3 Bone Defect Model 5.1.3 骨缺损模型
Bone defect is a condition in which the integrity of the bone structure is destroyed due to surgical trauma and more. The current models of bone defects mainly include cranial defects, long bone or segmental defect, partial cortical defect, and cancellous bone defect models. Models of bone defects are mostly obtained using animals by artificially destroying the bone.[104] If bone organoids are available, the microenvironment in human body can be perfectly mimicked with less loss of animals and lower cost.
骨缺损是由于手术创伤等原因导致骨骼结构完整性被破坏的情况。目前的骨缺损模型主要包括颅骨缺损、长骨或节段性缺损、部分皮质缺损和松质性骨缺损模型。骨缺损模型大多是用动物通过人工破坏骨骼获得的。104 如果有骨类器官，就可以完美地模拟人体的微环境，减少动物损失，降低成本。
5.1.4 Osteoarthritis Model
5.1.4 骨关节炎模型
Osteoarthritis is thought to be the most prevalent chronic degenerative joint disease, which is an age-related aseptic inflammatory disease.[105] As osteoarthritis has multiple etiologies, many modeling approaches are available, such as post-traumatic osteoarthritis caused by artificial trauma, or the corresponding models can be created by intra-articular cavity injection of drugs, gene knockout, and so on. It is possible to regulate the immune response of organoids and thereby establish a model of osteoarthritis. Organoid models of the bone-chondral junction interface have been designed by mechanical stimulation and different cell culture media to mimic the physiological conditions at the articulation.[106] This is simpler and more economical than the establishment of animal models.
骨关节炎被认为是最普遍的慢性退行性关节病，是一种与年龄相关的无菌性炎症性疾病。105 由于骨关节炎具有多种病因，因此有许多建模方法可用，例如人工创伤引起的创伤后骨关节炎，或者可以通过关节腔内注射药物、基因敲除等来创建相应的模型。可以调节类器官的免疫反应，从而建立骨关节炎模型。通过机械刺激和不同的细胞培养基设计了骨-软骨交界处的类器官模型，以模拟关节处的生理条件。106 这比建立动物模型更简单、更经济。
5.1.5 Genetic Bone Disease Model
5.1.5 遗传性骨病模型
Genetic bone disease refers to skeletal pathologies caused by developmental disorders due to genetic factors. Genetic disease models can be constructed in vitro by knocking out or editing the relevant genes using gene editing methods before introducing stem cells into organoid systems to mimic alterations in genetic material. In vitro models of genetic diseases in other organs have been established by constructing organoids. A study was completed by knocking out the GLA gene of iPSCs via gene editing and culturing into kidney organoids manifesting Fabry nephropathy for subsequent studies.[107] Another study explored the mechanism of congenital hypoplastic left heart syndrome by knocking out NKX2-5 and HANDS genes in cardiac organoids.[108] Combining gene editing technology with bone organoids enables the fabrication of bone genetic disease models such as congenital chondrodysplasia, congenital osteogenesis imperfecta, and Robinow syndrome, and to investigate their Pathogenesis and therapeutic approaches in vitro.
遗传性骨病是指由于遗传因素引起的发育障碍引起的骨骼病变。在将干细胞引入类器官系统以模拟遗传物质的改变之前，可以使用基因编辑方法敲除或编辑相关基因，从而在体外构建遗传疾病模型。通过构建类器官，已经建立了其他器官遗传病的体外模型。通过基因编辑敲除 iPSC 的 GLA 基因并培养成表现法布里肾病的肾脏类器官，用于后续研究，从而完成一项研究。107 另一项研究通过敲除心脏类器官中的 NKX2-5 和 HANDS 基因来探索先天性左心发育不良综合征的机制。108 将基因编辑技术与骨类器官相结合，可以构建骨遗传病模型，例如先天性软骨发育不良、先天性成骨不全症和 Robinow 综合征，并在体外研究它们的发病机制和治疗方法。
5.2 Bone Repair and Regeneration
5.2 骨骼修复和再生
The current clinical treatment for large bone defects is limited and poses many challenges for patients, such as the scarcity of bone tissue sources and severe rejection reactions. However, regenerative medicine has made it possible to repair tissue and organ damage in a more feasible and effective way.[109] Organoids, which are miniature organs grown in vitro, have emerged as a promising strategy for bone tissue regeneration as they offer a convenient and rejection-free autologous transplantation option. By deriving their own stem cells and inducing differentiation into bone in vitro, patients can obtain homologous bone that can be transplanted into the body for growth and development. Despite the numerous attempts made to construct bone organoids for bone regeneration, most current strategies are limited in their ability to mimic the complexity of bone structures and developmental processes. They often only mimic a certain stage or structure of bone, which is a single time and space mimicry.[110] This falls short of fully replicating the intricacies of bone formation and fails to achieve the necessary level of complexity required for clinical application. Therefore, there is an urgent need for new approaches to develop more advanced bone organoids that can closely resemble the intricate structures and functions of natural bone tissue. In conclusion, the establishment of organoids for bone tissue regeneration has significant potential for clinical application. However, current strategies must be refined and improved to more accurately mimic natural bone tissue in order to be effective. With further research and development, organoids have the potential to become a viable alternative for bone grafting and bone filler materials, offering patients a safer and more effective treatment option for large bone defects.
目前临床上对大骨缺损的治疗是有限的，给患者带来了许多挑战，例如骨组织来源的稀缺和严重的排斥反应。然而，再生医学使以更可行和有效的方式修复组织和器官损伤成为可能。109 类器官是在体外生长的微型器官，已成为一种很有前途的骨组织再生策略，因为它们提供了一种方便且无排斥反应的自体移植选择。通过获得自己的干细胞并在体外诱导分化为骨骼，患者可以获得可以移植到体内进行生长发育的同源骨骼。尽管人们多次尝试构建用于骨再生的骨类器官，但大多数当前策略在模拟骨骼结构和发育过程的复杂性方面的能力有限。它们通常只模仿骨骼的某个阶段或结构，这是单一的时空模拟。110 这不足以完全复制骨形成的复杂性，也未能达到临床应用所需的必要复杂性水平。因此，迫切需要新的方法来开发更先进的骨类器官，这些类器官可以紧密类似于天然骨组织的复杂结构和功能。综上所述，建立用于骨组织再生的类器官具有巨大的临床应用潜力。然而，当前的策略必须改进和改进，以更准确地模拟天然骨组织才能有效。 随着进一步的研究和开发，类器官有可能成为骨移植和骨填充材料的可行替代品，为患者提供更安全、更有效的大骨缺损治疗选择。
5.3 Drug Discovery and Toxicity Assessment
5.3 药物发现和毒性评估
Current drug discovery for human diseases is facing several limitations, such as the diversity of individual patients, unpredictable adverse outcomes, and time-consuming drug testing processes. While the use of organoids for drug discovery and screening has not been possible yet due to the complexity of constructing bone organoids, there have been successful applications of organoids in other fields.[100] One promising study involved the cultivation of mouse distal intestinal organoids in 96-well plates, where over 2000 pharmacologically active compounds were screened for inhibition of potassium ion transport. The study ultimately identified one of the most promising compounds for further investigation.[111] In addition, organoid models of primary liver cancer were also constructed, demonstrating that in vitro liver tumor organoids can express the same gene lineage as in vivo and possess the properties of primary liver cancer. This allows for high-throughput screening of oncology drugs on this model, potentially leading to the discovery of new treatments for liver cancer.[112] Therefore, the successful construction of bone organoids in the future would provide sufficient models for drug screening of emerging drugs for chronic diseases lacking clinical cases and reduce the time-consuming nature of drug testing. These advancements could lead to more effective treatments for a variety of illnesses and improve patient outcomes.
当前针对人类疾病的药物发现面临一些限制，例如个体患者的多样性、不可预测的不良结果以及耗时的药物测试过程。虽然由于构建骨类器官的复杂性，目前还无法将类器官用于药物发现和筛选，但类器官在其他领域已经成功应用。100 一项有前途的研究涉及在 96 孔板中培养小鼠远端肠道类器官，其中筛选了 2000 多种药理活性化合物以抑制钾离子转运。该研究最终确定了最有前途的化合物之一，以供进一步研究。111 此外，还构建了原发性肝癌的类器官模型，证明体外肝脏肿瘤类器官可以表达与体内相同的基因谱系，并具有原发性肝癌的特性。这允许在该模型上对肿瘤药物进行高通量筛选，从而有可能发现肝癌的新治疗方法。112 因此，未来骨类器官的成功构建将为缺乏临床病例的慢性疾病的新兴药物的药物筛选提供足够的模型，并减少药物检测的耗时性。这些进步可能会为各种疾病带来更有效的治疗方法，并改善患者的预后。
5.4 Personalized Medicine
5.4 个性化医疗
Precision medicine is an innovative approach that uses genetic information to diagnose and treat diseases in a personalized manner.[113] One of the techniques used is creating organoids from the patient's original cells, which possess the same genetic makeup as the patient. Organoids can be used to screen potential treatments and select drugs in vitro, reducing the risk of side effects during treatment. This method is particularly useful for tumors, which are known for their genetic heterogeneity. However, precision medicine can also be applied to bone-related primary and secondary tumors, as well as chronic conditions such as osteoarthritis and osteoporosis. By creating individual patients' organoids, precision medicine can provide tailored treatments that are specific to each patient's unique genetic makeup, leading to more effective outcomes and improved quality of life. The creation of organoids from a patient's original cells is a cutting-edge technique that has shown promising results in precision medicine. Organoids can mimic the behavior of organs in the human body, making them an ideal tool for drug testing and personalized treatment. As tumors have genetic variations from person to person, organoids derived from a patient's tumor can be used to screen potential treatments and select the best drugs for that particular patient, increasing the likelihood of a successful outcome.[4] Moreover, this technique can also be applied to bone-related primary and secondary tumors, which are notoriously difficult to treat. Precision medicine is not limited to cancer as it can also be used to explore the effects of different genes and perform drug screening for chronic conditions such as osteoarthritis and osteoporosis. By creating organoids that possess the same genetic makeup as the patient, researchers can test potential treatments in vitro, avoiding the risk of adverse side effects that can occur during clinical trials. This approach provides a safer and more efficient way to identify effective drugs and personalized treatments for patients. The creation of organoids from a patient's original cells is an exciting technique that has shown tremendous potential in the treatment of various diseases, including cancer and bone-related conditions. With the continued advancement of precision medicine, we can expect to see more effective treatments, improved patient outcomes; and ultimately, a better quality of life for patients.[114
精准医疗是一种创新方法，它使用遗传信息以个性化的方式诊断和治疗疾病。113 使用的技术之一是从患者的原始细胞中产生类器官，这些细胞具有与患者相同的基因组成。类器官可用于筛选潜在的治疗方法和体外选择药物，从而降低治疗期间出现副作用的风险。这种方法对肿瘤特别有用，因为肿瘤以其遗传异质性而闻名。然而，精准医学也可以应用于与骨骼相关的原发性和继发性肿瘤，以及骨关节炎和骨质疏松症等慢性病。通过创建个体患者的类器官，精准医学可以针对每个患者独特的基因构成提供量身定制的治疗，从而带来更有效的结果和更好的生活质量。从患者的原始细胞中创造类器官是一项尖端技术，在精准医学中已显示出可喜的成果。类器官可以模拟人体器官的行为，使其成为药物检测和个性化治疗的理想工具。由于肿瘤因人而异，因此源自患者肿瘤的类器官可用于筛选潜在的治疗方法并为该特定患者选择最佳药物，从而增加成功结果的可能性。4 此外，这项技术还可以应用于与骨相关的原发性和继发性肿瘤，这些肿瘤是众所周知的难以治疗。精准医学不仅限于癌症，它还可用于探索不同基因的影响，并对骨关节炎和骨质疏松症等慢性病进行药物筛选。 通过创造与患者具有相同基因组成的类器官，研究人员可以在体外测试潜在的治疗方法，避免临床试验期间可能发生的不良副作用的风险。这种方法提供了一种更安全、更有效的方式来识别有效的药物和为患者提供个性化治疗。从患者的原始细胞中创造类器官是一项令人兴奋的技术，在治疗各种疾病（包括癌症和骨骼相关疾病）方面显示出巨大的潜力。随着精准医疗的不断发展，我们可以期待看到更有效的治疗方法，改善患者预后;并最终提高患者的生活质量。114 元]
5.5 Developmental Biology Research
5.5 发育生物学研究
Developmental biology is a field of study that delves into the intricate processes that occur during an organism's growth and development, from the cellular to the molecular level. With the use of organoids—artificially grown organ-like structures—from undifferentiated stem cells, scientists can investigate the generation and development of tissues and organs within a microenvironment that mimics the conditions of a living organism. For example, bone organoids can recreate the process of osteogenesis (the formation of bone tissue) as it occurs in living organisms, including endochondral and intramembranous osteogenesis. By examining the differential expression of genes and proteins during osteogenesis, researchers can gain insights into the molecular mechanisms involved in bone development and growth. Overall, organoids provide a powerful tool for investigating the complex processes that underlie the development of living organisms (Figure 7).
发育生物学是一个研究领域，深入研究生物体生长和发育过程中发生的从细胞到分子水平的复杂过程。通过使用来自未分化干细胞的类器官（人工生长的器官样结构），科学家可以在模拟生物体条件的微环境中研究组织和器官的产生和发育。例如，骨类器官可以重现生物体中发生的成骨过程（骨组织的形成），包括软骨内和膜内成骨。通过检查成骨过程中基因和蛋白质的差异表达，研究人员可以深入了解骨骼发育和生长所涉及的分子机制。总体而言，类器官为研究生物体发育的复杂过程提供了强大的工具（图7）。
骨类器官的应用。骨类器官可应用于疾病建模、骨再生、药物发现、骨相关药物的毒性测试、精准医学的基因相关疗法和发育生物学研究。
6 Prospects and Challenges
6 前景与挑战
From basic research to clinical applications, bone organoids hold wide promise. Spatial and temporal regulation of the cellular differentiation and ECM mimetic materials will continue to be focus of future research. However, there are still some challenges and obstacles to be solved in the development of bone organoids (Figure 8).
从基础研究到临床应用，骨类器官前景广阔。细胞分化和 ECM 模拟材料的时空调控将继续成为未来研究的重点。然而，在骨类器官的发展中仍存在一些挑战和障碍需要解决（图8）。
骨类器官的未来挑战。骨类器官的未来挑战在于四个主要领域：复杂性、血管形成、标准化和多器官通信。使用 BioRender.com 创建。
6.1 Complexity 6.1 复杂性
Bone tissue is a complex and dynamic structure that performs several essential functions, such as support, protection, and hematopoiesis, requiring diverse structures at different times. Thus, developing functional bone organoids that replicate this complexity is a significant challenge. The appropriate levels of mineralization, cell type diversity, and extracellular matrix (ECM) composition and organization are crucial for achieving this goal. To construct a perfect bone organoid, the ECM must meet the condition of spatially forming a sequential structure and temporally performing different functions according to physiological requirements. One study demonstrated the regulation of cellular structure by ECM by using a two-layer porcine-derived bone scaffold consisting of cancellous and cortical bone to mimic ECM and to promote the formation of cortical and trabecular bone structures.[115] Although current tissue engineering techniques have made significant contributions to this direction, achieving multi-layered ECM synthesis may be possible in the future using advanced technologies such as 3D printing.
骨组织是一种复杂而动态的结构，它执行多种基本功能，例如支持、保护和造血，在不同时期需要不同的结构。因此，开发复制这种复杂性的功能性骨类器官是一项重大挑战。适当水平的矿化、细胞类型多样性以及细胞外基质 （ECM） 组成和组织对于实现这一目标至关重要。为了构建完美的骨类器官，ECM 必须满足在空间上形成序列结构并根据生理需求在时间上执行不同功能的条件。一项研究表明，ECM 通过使用由松质骨和皮质骨组成的两层猪衍生骨支架来模拟 ECM 并促进皮质和小梁骨结构的形成，从而调节细胞结构。115 尽管当前的组织工程技术对这一方向做出了重大贡献，但未来使用 3D 打印等先进技术可能实现多层 ECM 合成。
6.2 Vascularization 6.2 血管形成
The creation of bone organoids presents a significant challenge in the field of tissue engineering, namely the issue of vascularization.[116] The intricate blood supply of bone is comprised of periosteal vessels and trophoblastic vessels, which intricately branch into capillaries throughout the osteon lamella and Haversian canals, providing essential nutritional support for bone growth and development. While current techniques for constructing bone organoids rely on artificial nutrition supplies in vitro, such methods may prove insufficient when applied to the repair of bone defects, thereby making vascularization an unavoidable problem.[117] Indeed, this is a concern faced by many other types of organoids as well. To overcome this obstacle, researchers have begun exploring the co-culturing of endothelial cells and stem cells on a 3D matrix, which may enable the construction of organoids containing blood vessels.[118] A research team has also developed self-organized 3D human vascular organoids by inducing pluripotent stem cell differentiation to form complete and reproducible vascular systems. Future challenges in vascularization of bone organoids could also be solved using in vitro construction and assembly of vascular organoids (Figure 9A).[119] Vascularization is not only a problem to be solved in the construction of bone organoids but also a general challenge in the construction of other organoids. In the construction of brain organoids, a research team has been able to differentiate some stem cells into vascular endothelial cells by transfecting the ETV2 transcription factor and adjusting the differentiation conditions of hESC, resulting in the construction of a functional vascular system and also improving the maturation of brain organoids (Figure 9B).[120] Another research team designed a microfluidic culture system to dynamically co-culture primary human endothelial cells and hPSCs to construct kidney organoids and verified that dynamic culture can promote the process of organoid vascularization in vitro (Figure 9C).[121
骨类器官的创建在组织工程领域提出了一个重大挑战，即血管形成问题。116 骨骼错综复杂的血液供应由骨膜血管和滋养细胞血管组成，它们错综复杂地分支成整个骨板和 Haversian 管的毛细血管，为骨骼生长和发育提供必要的营养支持。虽然目前构建骨类器官的技术依赖于体外的人工营养供应，但当应用于修复骨缺损时，这些方法可能被证明是不够的，从而使血管形成成为一个不可避免的问题。117 事实上，这也是许多其他类型的类器官面临的问题。为了克服这一障碍，研究人员已经开始探索内皮细胞和干细胞在 3D 基质上的共培养，这可能能够构建包含血管的类器官。118 一个研究团队还通过诱导多能干细胞分化以形成完整且可重复的血管系统，开发了自组织的 3D 人类血管类器官。骨类器官血管化的未来挑战也可以使用血管类器官的体外构建和组装来解决（图9A）。119 血管化不仅是骨类器官构建中需要解决的问题，也是其他类器官构建中的普遍挑战。 在脑类器官的构建中，一个研究团队已经能够通过转染 ETV2 转录因子和调整 hESC 的分化条件，将一些干细胞分化为血管内皮细胞，从而构建功能性血管系统，也促进了脑类器官的成熟（图 9B）。120 另一个研究小组设计了一种微流控培养系统，以动态共培养原代人内皮细胞和 hPSC 以构建肾类器官，并验证动态培养可以促进体外类器官血管化的过程（图 9C）。121 元]
类器官血管化的挑战。A） 血管类器官的体外构建。经许可转载。119 版权所有 2019，施普林格自然。B） 通过用转录因子 ETV2 转染 hESC 并改变培养条件来促进脑类器官的血管化。血管形成程度可以通过大体形态和荧光染色的变化来观察。经许可转载。120 版权所有 2023，施普林格自然。C） 动态培养系统促进肾脏类器官的体外血管化。体外血管形成通过荧光染色显示。经许可转载。121 版权所有 2023，施普林格自然。
6.3 Standardization 6.3 标准化
Bone organoids are a novel field of research that is still in its early stages, and as such, there are currently no standardized raw materials or procedures in place. This lack of standardization is a significant challenge when it comes to manufacturing bone organoids in bulk as the heterogeneity of the various cell types and external matrix types used to construct bone organoids can vary significantly from one experiment to another. As a result, the reproducibility of bone organoids is poor, making it difficult for researchers to compare results and draw meaningful conclusions from their studies. Despite these challenges, researchers continue to explore new approaches to the construction of bone organoids, with the hope of one day developing standardized methods that will allow for more consistent and reproducible results.
骨类器官是一个仍处于早期阶段的新兴研究领域，因此，目前没有标准化的原材料或程序。在批量生产骨类器官时，缺乏标准化是一个重大挑战，因为用于构建骨类器官的各种细胞类型和外部基质类型的异质性可能因实验而异。因此，骨类器官的可重复性很差，使研究人员难以比较结果并从研究中得出有意义的结论。尽管存在这些挑战，研究人员仍在继续探索构建骨类器官的新方法，希望有一天能开发出标准化的方法，从而获得更一致和可重复的结果。
6.4 Multi-Organ Communication
6.4 多器官交流
Currently, bone organoids are limited in their ability to accurately simulate the pathological changes of multiple organs. This is because diseases related to bone not only affect the bone tissue itself but also involve other bodily systems such as the nervous and muscular systems. For example, hormone levels in the body can impact the balance of bone remodeling, and the nervous system also plays a regulatory role in this process. However, advancements in organ-on-a-chip technology have allowed for the simulation of these multi-organ associations at the cellular level. One study has reproduced interdependent organ functions by using tissue chips to simulate mature human heart, liver, bone and skin tissue niches connected by recirculating vascular flow.[122] In the future, it may be possible to create more comprehensive multi-organ organoid models using various strategies such as promoting stem cell differentiation or co-culturing different cell types in vitro. This could greatly enhance our understanding of complex diseases and lead to more effective treatments.
目前，骨类器官在准确模拟多个器官的病理变化的能力方面受到限制。这是因为与骨骼相关的疾病不仅影响骨组织本身，还涉及其他身体系统，例如神经和肌肉系统。例如，体内的激素水平会影响骨骼重塑的平衡，神经系统在此过程中也起着调节作用。然而，器官芯片技术的进步允许在细胞水平上模拟这些多器官关联。一项研究通过使用组织芯片来模拟由循环血管流连接的成熟人类心脏、肝脏、骨骼和皮肤组织生态位，从而再现了相互依赖的器官功能。122 未来，有可能使用各种策略创建更全面的多器官类器官模型，例如促进干细胞分化或在体外共培养不同的细胞类型。这可以大大增强我们对复杂疾病的理解，并带来更有效的治疗方法。
6.5 Other Challenges 6.5 其他挑战
Bone, being a crucial structure that bears mechanical stresses in our bodies, must constantly adapt to its functions through a process of stress modulation and micro-modification under normal physiological conditions. However, bone organoids face a unique challenge compared to other types of organoids. This challenge arises from the distinct developmental timeline of bone tissue. In nature, bone development and mineralization are processes that demand patience. It can take anywhere from one to several months in vivo for a bone defect to heal and form a bone callus,[123] and in laboratory experiments, the mineralization process often extends over a span of 10 days to several months.[124] This extended formation period presents the specific requirement that the cells used in bone organoid construction must maintain their capacity to differentiate and proliferate over an extended duration. Addressing this issue may also involve innovative material design to support the sustained development of bone organoids.[125] A study prepared gelatin-based structurally dynamic hydrogel (GelCD hydrogel) that emulated the intrinsic structural dynamics of the ECM, promoted clonal expansion of mouse embryonic stem cells (mESCs) cultured in 3D, and better maintained the proliferative differentiation capacity of the embryonic stem cells in the hydrogel within 2 months.[126] It might also be possible to reduce the time to mineralization by accelerating the mineralization factor. It is believed that this class of problems can be properly addressed in the construction of different types of bone organoids.
骨骼作为我们体内承受机械应力的关键结构，在正常生理条件下，必须通过应力调节和微修饰过程不断适应其功能。然而，与其他类型的类器官相比，骨类器官面临着独特的挑战。这一挑战源于骨组织独特的发育时间表。在自然界中，骨骼发育和矿化是需要耐心的过程。在体内，骨缺损可能需要一到几个月的时间才能愈合并形成骨愈伤组织，123 在实验室实验中，矿化过程通常会持续 10 天到几个月。124 这种延长的形成期提出了一个具体要求，即用于骨类器官构建的细胞必须在较长时间内保持其分化和增殖的能力。解决这个问题还可能涉及创新的材料设计，以支持骨类器官的可持续发展。125 一项研究制备了基于明胶的结构动态水凝胶 （GelCD 水凝胶），该水凝胶模拟 ECM 的内在结构动力学，促进了 3D 培养的小鼠胚胎干细胞 （mESC） 的克隆扩增，并在 2 个月内更好地维持了胚胎干细胞在水凝胶中的增殖分化能力。126 也可以通过加速矿化因子来缩短矿化时间。据信，在构建不同类型的骨类器官时可以适当地解决这类问题。
In conclusion, the emergence of bone organoids is of great significance to the research of biomedical and clinical applications. The research of bone organoids remains in the primary exploration stage, and continuous research will provide more perspectives and progress on the construction of bone organoids in the future.
综上所述，骨类器官的出现对生物医学和临床应用的研究具有重要意义。骨类器官的研究仍处于初级探索阶段，持续的研究将为未来骨类器官的构建提供更多的视角和进展。
Acknowledgements 确认
D.Z and Q.S contributed equally to this work. W.C is the main author. This work was supported by grants from the National Key Research and Development Program of China (2020YFA0908200), the National Nature Science Foundation of China (52273133), the Shanghai Municipal Health and Family Planning Commission (2022XD055).
D.Z 和 Q.S 对这项工作的贡献相同。W.C 是主要作者。这项工作得到了中国国家重点研发计划 （2020YFA0908200）、中国国家自然科学基金 （52273133）、上海市卫生和计划生育委员会 （2022XD055） 的资助。
Conflict of Interest 利益冲突
The authors declare no conflict of interest.
作者声明没有利益冲突。
Biographies 列传
Ding Zhao is a current Master's candidate in Orthopedic Surgery at the Ruijin Hospital, Shanghai Jiaotong University School of Medicine. His research is dedicated to regenerative biomaterials and innovative drug delivery systems for the skeletal system.
Ding Zhao 是上海交通大学医学院附属瑞金医院骨外科硕士研究生。他的研究致力于骨骼系统的再生生物材料和创新药物递送系统。
Qimanguli Saiding is currently a Ph.D. student at the Shanghai Jiao Tong University School of Medicine affiliated Ruijin Hospital, specializing in surgery. She started her academic journey primarily focusing on the development and design of anti-inflammatory reconstructive biomaterials.
Qimanguli Saiding 目前是上海交通大学医学院附属瑞金医院的一名博士生，专攻外科。她的学术之旅开始主要集中在抗炎重建生物材料的开发和设计上。
Wenguo Cui is a full professor at the Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. Currently, he is Fellow of the Royal Society of Chemistry and Fellow of Chinese Society of Biomaterials. His scientific interests are focused on the development of novel biomaterials and nanomaterials for tissue regeneration, drug delivery, and disease treatment.
崔文国是上海交通大学医学院附属瑞金医院的正教授。目前，他是英国皇家化学会会士和中国生物材料学会会士。他的科学兴趣集中在开发用于组织再生、药物输送和疾病治疗的新型生物材料和纳米材料。
